Compositions and methods for differentiating stem cells into nk cells

ABSTRACT

The disclosure features methods and compositions for differentiating stem cells into hematopoietic stem and progenitor cells (HSPC) and/or Natural Killer (NK) cells. The methods and compositions described herein are used to differentiate stem or progenitor cells having at least one gene-edit that is maintained in the differentiated cell. Also provided are differentiated cells produced using the methods and compositions described herein for therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.63/132,230 filed Dec. 30, 2020 and U.S. Provisional Application No.63/250,037, filed Sep. 29, 2021, the entire contents of which are herebyincorporated by reference.

FIELD

The invention relates to methods and compositions for differentiatingstem cells into hematopoietic stem and progenitor cells (HSPC) and/orNatural Killer (NK) cells.

BACKGROUND

Natural Killer (NK) cells are lymphocytes involved in the innate immuneresponse. Due to their function, NK cells are becoming cells of interestfor use in the treatment of different diseases such as cancer. Recentsuccess in editing immune cells (e.g. CAR T cells) for enhancedtherapeutic ability prompts the use of NK cells in further therapydiscoveries. Unfortunately, differentiating natural killer cells istypically a low output 5 to 6-week process. Additionally, currentmethods require feeder cells and cell sorting which adds additional timeand cost for generating the cells. Accordingly, methods ofdifferentiation are needed that reduce the cost, increase cell output,and reduce the time needed to generate NK cells. Improving upon thesemethods will allow for efficient output of NK cells and NK cell therapyfor use in treating disease.

SUMMARY OF THE INVENTION

In some aspects, the disclosure provides a method for generating NaturalKiller (NK) cells from stem cells, the method comprising: (a) culturinga population of stem cells in a first medium comprising a ROCK inhibitorunder conditions sufficient to form aggregates; (b) culturing theaggregates in a second medium comprising BMP-4; (c) culturing theaggregates in a third medium comprising BMP-4, FGF2, a WNT pathwayactivator, and Activin A; (d) culturing the aggregates in a fourthmedium comprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and anactivin/nodal inhibitor to form a cell population comprisinghematopoietic stem and progenitor cells (HSPCs); (e) culturing the cellpopulation in a fifth medium comprising FGF2, VEGF, TPO, SCF, IL-3 andFLT3L; (f) culturing the cell population in a sixth medium comprisingIL-3, IL-7, FLT3L, IL-15 and SCF; and (g) culturing the cell populationin a seventh medium comprising IL-7, FLT3L, IL-15 and SCF for a timesufficient to generate NK cells.

In some aspects, the disclosure provides a method for generating NaturalKiller (NK) cells from stem cells, the method comprising: (a) culturinga population of stem cells in a first medium comprising a ROCK inhibitorunder conditions sufficient to form aggregates; (b) culturing theaggregates in a second medium comprising BMP-4; (c) culturing theaggregates in a third medium comprising BMP-4, FGF2, a WNT pathwayactivator, and Activin A; (d) culturing the aggregates in a fourthmedium comprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and anactivin/nodal inhibitor to form a cell population comprisinghematopoietic stem and progenitor cells (HSPCs); (e) culturing the cellpopulation in a fifth medium comprising FGF2, VEGF, TPO, SCF, IL-3 andFLT3L; (f) culturing the cell population in a sixth medium comprisingIL-3, IL-7, FLT3L, IL-15 and SCF; and (g) culturing the cell populationin a seventh medium comprising IL-7, FLT3L, IL-15 and SCF; and (h)culturing the cell population in an eighth medium comprising IL-7,FLT3L, IL-15, SCF and nicotinamide for a time sufficient to generate NKcells.

In some aspects, the disclosure provides a method for generating NaturalKiller (NK) cells from stem cells, the method comprising: (a) culturinga population of stem cells in a first medium comprising a ROCK inhibitorunder conditions sufficient to form aggregates; (b) culturing theaggregates in a second medium comprising BMP-4; (c) culturing theaggregates in a third medium comprising BMP-4, FGF2, a WNT pathwayactivator, and Activin A; (d) culturing the aggregates in a fourthmedium comprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and anactivin/nodal inhibitor to form a cell population comprisinghematopoietic stem and progenitor cells (HSPCs); (e) culturing the cellpopulation in a fifth medium comprising FGF2, VEGF, TPO, SCF, IL-3 andFLT3L; (f) culturing the cell population in a sixth medium comprisingIL-3, IL-7, FLT3L, IL-15 and SCF; and (g) culturing the cell populationin a seventh medium comprising IL-7, FLT3L, IL-15 and SCF; and (h)culturing the cell population in an eighth medium comprising IL-7,FLT3L, IL-15, and SCF for a time sufficient to generate NK cells.

In some aspects, culturing the cell population in the fifth medium instep (e) results in the cell population comprising at least about 25% ofHSPCs, optionally comprising about 25% to about 55% of HSPCs. In someaspects, culturing the cell population in the fifth medium in step (e)results in the cell population comprising about 29% to about 50% ofHSPCs. In some aspects, culturing the cell population in the fifthmedium in step (e) results in the cell population comprising about 36%of HSPCs or about 50% of HSPCs.

In some aspects, culturing the cell population in the sixth medium instep (f) results in the formation of progenitor cell populationcomprising common lymphoid progenitor (CLP) cells. In some aspects, theprogenitor cell population comprises at least about 15% of CLP cells,optionally wherein the CLP cells express CD7 and CD45. In some aspects,the progenitor cell population comprises about 15% to about 50% of CLPcells, optionally about 19% to about 45%. In some aspects, theprogenitor cell population comprises about 35% of CLP cells.

In some aspects, culturing the cell population in the seventh medium instep (g) results in the cell population comprising at least about 70% ofNK cells. In some aspects, culturing the cell population in the seventhmedium in step (g) results in the cell population comprising at leastabout 95% of NK cells.

In some aspects, culturing the cell population in the eighth medium instep (h) results in the cell population comprising at least about 70% ofNK cells. In some aspects, culturing the cell population in the eighthmedium in step (h) results in the cell population comprising at leastabout 95% of NK cells.

In some aspects, the second medium further comprises a ROCK inhibitor.In some aspects, the ROCK inhibitor is thiazovivin. In some aspects, theROCK inhibitor is Y27632.

In some aspects, the WNT pathway activator is CHIR-99021. In someaspects, the activin/nodal inhibitor is SB-431542.

In some aspects, step (a) comprises culturing for 12-48 hours. In someaspects, step (b) comprises culturing for up to 24 hours. In someaspects, step (c) comprises culturing for 1-3 days. In some aspects,step (d) comprises culturing for 1-3 days. In some aspects, step (e)comprises culturing for 1-3 days. In some aspects, step (f) comprisesculturing for at least 6 days and up to 8 days. In some aspects, step(g) comprises culturing for at least 6 days and up to 21-28 days total.In some aspects, step (g) comprises culturing for up to 6 days and step(h) comprises culturing for at least 6 days and up to 10-16 days total.

In any of the foregoing or related aspects, step (a) comprises culturingfor 16-20 hours; step (b) comprises culturing for 6-10 hours; step (c)comprises culturing for 2 days; step (d) comprises culturing for 2 days;step (e) comprises culturing for 2 days; step (f) comprises culturingfor 6-8 days; and step (g) comprises culturing for 6-28 days.

In any of the foregoing or related aspects, step (a) comprises culturingfor 16-20 hours; step (b) comprises culturing for 6-10 hours; step (c)comprises culturing for 2 days; step (d) comprises culturing for 2 days;step (e) comprises culturing for 2 days; step (f) comprises culturingfor 6-8 days; step (g) comprises culturing for 6 days; and step (h)comprises culturing for 6-16 days.

In some aspects, steps (a)-(g) occurs between 20-42 days. In someaspects, steps (a)-(g) occurs in less than 20 days. In some aspects, NKcells are generated in about 20 days. In some aspects, steps (a)-(g)occurs in about 20 days and culturing the cell population in the seventhmedium in step (g) results in the cell population comprising at leastabout 70% NK cells or 95% NK cells.

In some aspects, steps (a)-(h) occurs between 19-33 days. In someaspects, steps (a)-(h) occurs in less than 20 days. In some aspects, NKcells are generated in about 20 days. In some aspects, NK cells aregenerated in about 16 days. In some aspects, NK cells are generated inabout 23 to 40 days. In some aspects, steps (a)-(h) occurs in about23-40 days. In some aspects, NK cells are generated in about 23 to 30days. In some aspects, steps (a)-(h) occurs in about 23-30 days. In someaspects, steps (a)-(h) occurs in about 28-30 days. In some aspects,culturing the cell population in the eighth medium in step (h) resultsin the cell population comprising at least about 70% NK cells or 95% NKcells. In some aspects, steps (a)-(h) occurs in about 30 days andculturing the cell population in the eighth medium in step (h) resultsin the cell population comprising at least about 70% NK cells or 95% NKcells.

In any of the foregoing or related aspects, the method is carried outunder suspension agitation. In some aspects, suspension agitationcomprises rotation, optionally wherein the rotation speed is at leastabout 35 RPM to about 100 RPM.

In any of the foregoing or related aspects, the first and second mediacomprise StemFlex medium. In any of the foregoing or related aspects,the first media comprises StemFlex medium or StemBrew medium. In someaspects, the third, fourth and fifth media comprise APEL medium. In someaspects, the second, third, fourth and fifth media comprise APEL medium.In some aspects, the sixth and seventh media comprise DMEM/F12 medium.In some aspects, the sixth and seventh media comprise DMEM (highglucose)/F12 medium.

In any of the foregoing or related aspects, the sixth and seventh mediacomprise human serum, zinc sulfate, ethanolamine, β-mercaptoethanol,glucose, or any combination thereof. In some aspects, the concentrationof human serum is about 5%-40%, the concentration of zinc sulfate isabout 1.7-40 μM, the concentration of ethanolamine is about 20-60 μM,the concentration of β-mercaptoethanol is about 0.5-45 μM, and theconcentration of glucose is about 8-40 mM. In some aspects, theconcentration of human serum is about 15%, the concentration of zincsulfate is about 37 μM, the concentration of ethanolamine is about 50μM, the concentration of β-mercaptoethanol is about 1 μM, and theconcentration of glucose is about 27 mM. In some aspects, theconcentration of human serum is about 20%, the concentration of zincsulfate is about 36.2 μM, the concentration of ethanolamine is about 50μM, and the concentration of glucose is about 20 mM. In some aspects,the sixth and seventh media do not comprise β-mercaptoethanol.

In any of the foregoing or related aspects, the sixth and seventh mediacomprises DMEM/F12 medium and a supplement of human serum, zinc sulfate,ethanolamine, β-mercaptoethanol, glucose, or any combination thereof. Inany of the foregoing or related aspects, the sixth and seventh mediacomprises DMEM (high glucose)/F12 medium and a supplement of humanserum, zinc sulfate, ethanolamine, β-mercaptoethanol, glucose, or anycombination thereof. In some aspects, the supplement provides anadditional concentration of human serum of about 5%-40%, an additionalconcentration of zinc sulfate of about 1.7-40 μM, an additionalconcentration of ethanolamine of about 20-60 μM, an additionalconcentration of s-mercaptoethanol of about 0.5-45 μM, and an additionalconcentration of glucose of about 2-40 mM. In some aspects, theadditional concentration of human serum is about 15%, the additionalconcentration of zinc sulfate is about 37 μM, the additionalconcentration of ethanolamine is about 50 μM, the additionalconcentration of β-mercaptoethanol is about 1 μM, and the additionalconcentration of glucose is about 27 mM. In some aspects, the additionalconcentration of human serum is about 15%, the additional concentrationof zinc sulfate is about 37 μM, the additional concentration ofethanolamine is about 50 AM, the additional concentration ofβ-mercaptoethanol is about 1 μM, and the additional concentration ofglucose is about 10.25 mM. In some aspects, the additional concentrationof human serum is about 20%, the additional concentration of zincsulfate is about 36.2 μM, the additional concentration of ethanolamineis about 50 μM, and the additional concentration of glucose is about 20mM. In some aspects, the additional concentration of human serum isabout 20%, the additional concentration of zinc sulfate is about 36.2μM, the additional concentration of ethanolamine is about 50 μM, and theadditional concentration of glucose is about 4.66 mM. In some aspects,the sixth and seventh media do not comprise a supplement comprisingβ-mercaptoethanol.

In any of the foregoing or related aspects, the eighth media comprisesDMEM/F12 or DMEM (high glucose)/F12 medium and a supplement of humanserum, zinc sulfate, ethanolamine, glucose, or any combination thereof.In some aspects, the supplement provides an additional concentration ofhuman serum of about 5%-40%, an additional concentration of zinc sulfateof about 1.7-40 AM, an additional concentration of ethanolamine of about20-60 μM, and an additional concentration of glucose of about 2-40 mM.In some aspects, the additional concentration of human serum is about10%, the additional concentration of zinc sulfate is about 37 μM, theadditional concentration of ethanolamine is about 50 AM, and theadditional concentration of glucose is about 2.3 mM.

In some aspects, the first medium comprises 10 μM of the ROCK inhibitor.In some aspects, the second medium comprises 30 ng/mL BMP-4. In someaspects, the second medium further comprises 10 μM of a ROCK inhibitor.In some aspects, the second medium comprises 30 ng/mL BMP-4 and 10 μM ofa ROCK inhibitor. In some aspects, the third medium comprises 30 ng/mLBMP-4, 100 ng/mL FGF2, 3-10 μM CHIR-99021, optionally 6 μM CHIR-99021 or7 μM CHIR-99021, and 2.5-5.0 ng/mL Activin A. In some aspects, the thirdmedium is added to the second medium at a 1:1 ratio. In some aspects,the fourth and fifth media comprise 20 ng/mL FGF, 20 ng/mL VEGF, 20ng/mL TPO, 100 ng/mL SCF, 40 ng/mL IL-3, and 10-20 ng/mL FLT3L. In someaspects, the fourth medium further comprises 5 μM SB-431542. In someaspects, the fourth medium further comprises 0.5-5 μM WNT C-59. In someaspects, the sixth and seventh media comprises 20 ng/mL IL-7, 10-20ng/mL FLT3L, 10-20 ng/mL IL-15, and 20 ng/mL SCF. In some aspects, thesixth medium comprises 5 ng/mL IL-3.

In any of the foregoing or related aspects, the eighth medium cancomprise IL-7, FLT3L, IL-15, SCF and nicotinamide. In various aspects,the eighth medium can comprise 10-20 ng/mL IL-7, 5-20 ng/mL FLT3L, 10-30ng/mL IL-15, 20-40 ng/mL SCF, and 1-15 mM nicotinamide. In variousaspects, the eighth medium comprises 10 ng/mL IL-7, 7.5 ng/mL FLT3L, 15ng/mL IL-15, 20 ng/mL SCF and 6.5 mM nicotinamide.

In any of the foregoing or related aspects, the eighth medium cancomprise IL-7, FLT3L, IL-15, and SCF. In various aspects, the eighthmedium can comprise 10-20 ng/mL IL-7, 5-20 ng/mL FLT3L, 10-30 ng/mLIL-15, and about 20-40 ng/mL SCF. In various aspects, the eighth mediumcomprises about 10 ng/mL IL-7, about 7.5 ng/mL FLT3L, about 15 ng/mLIL-15, and about 20 ng/mL SCF. In various aspects, the eighth mediumdoes not comprise nicotinamide.

In any of the foregoing or related aspects, the HSPCs of (d) expressCD34 and/or CD45. In some aspects, the NK cells express CD56 and/orCD45. In some aspects, the NK cells express at least one activatingreceptor. In some aspects, the at least one activating receptor isselected from the group of NKp44, NKp46, NKG2D, CD16, KIR2DL4, NKp30,and any combination thereof. In some aspects, the NK cells express atleast one inhibitory receptor. In some aspects, the at least oneinhibitory receptor is selected from the group of NKG2A, KIR3DL2, andany combination thereof. In some aspects, the NK cells express at leastone co-receptor. In some aspects, the at least one co-receptor is CD94.In some aspects, the NK cells comprise at least one function associatedwith endogenous NK cells. In some aspects, the at least one functioncomprises the ability to induce cell lysis and cell death of a targetcell. In some aspects, the at least one function comprisesdegranulation. In some aspects, degranulation comprises release ofperforin and granzyme B. In some aspects, degranulation comprisesexpression of CD107a on the cell surface of an NK cell.

In some aspects, the NK cells are generated without sorting CD34+ cellsfrom the cell population.

In some aspects, the population of stem cells is a population ofengineered cells. In some aspects, the stem cells are geneticallymodified by an RNA-guided endonuclease system. In some aspects, theRNA-guided endonuclease system is a CRISPR system comprising a CRISPRnuclease and a guide RNA.

In any of the foregoing or related aspects, the stem cells are inducedpluripotent stem cells (iPSC), pluripotent stem cells (PSC), embryonicstem cells (ESC), or adult stem cells (ASC). In some aspects, the stemcell is a mammalian cell, optionally wherein the cell is a human cell.

In some aspects, the disclosure provides a population of stem cellsdifferentiated by or obtainable by a method described herein. In someaspects, the disclosure provides a population of hematopoietic stem andprogenitor cells differentiated by or obtainable by at least one step ina method described herein. In other aspects, the disclosure provides aplurality of NK cells generated by or obtainable by a method describedherein.

In some aspects, the disclosure provides a composition comprising aplurality of NK cells generated by or obtainable by a method describedherein, for use as a medicament. In other aspects, the disclosureprovides a composition comprising a population of stem cells (e.g.,hematopoietic stem and progenitor cells) differentiated by or obtainableby a method described herein, for use as a medicament. In some aspects,the composition may be a pharmaceutical composition.

In some aspects, the population of cells and/or the composition providedherein is provided for the use of treating a subject in need thereof(e.g., treating a condition in a subject in need thereof). In someaspects, the plurality of NK cells, the population of stem cells (e.g.,hematopoietic stem and progenitor cells) and/or the composition providedherein is provided for the use of treating a subject in need thereof(e.g., treating a condition in a subject in need thereof). In someaspects, the disclosure provides a plurality of NK cells for use intreating a subject in need thereof. In some aspects, the disclosureprovides a population of hematopoietic stem and progenitor cells for usein treating a subject in need thereof. In some aspects, the subject is ahuman who has, is suspected of having, or is at risk for a cancer. Insome aspects, the subject is a human who has, is suspected of having, oris at risk for an infectious disease or an autoimmune disease. In someaspects, the plurality of NK cells, the population of stem cells (e.g.,hematopoietic stem and progenitor cells) and/or the composition providedherein is provided for the use for treating cancer. In some aspects, theplurality of NK cells, the population of stem cells (e.g., hematopoieticstem and progenitor cells) and/or the composition provided herein isprovided for the use for treating an infectious disease or an autoimmunedisease.

In other aspects, the disclosure provides a method comprisingadministering to a subject a plurality of NK cells described herein or apharmaceutical composition comprising the plurality of NK cellsdescribed herein. In some aspects, the plurality of NK cells isadministered as a pharmaceutical composition. In other aspects, thedisclosure provides a method comprising administering to a subject apopulation of hematopoietic stem and progenitor cells described herein.In some aspects, the population of hematopoietic stem and progenitorcells is administered as a pharmaceutical composition. In some aspects,the subject is a human who has, is suspected of having, or is at riskfor a cancer. In some aspects, the subject is a human who has, issuspected of having, or is at risk for an infectious disease or anautoimmune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic comparison of a published iNK (NK cellsdifferentiated from iPSC) differentiation protocol and an exemplarymodified iNK differentiation protocol described herein.

FIG. 2A provides a schematic timeline and cell stages of iNKdifferentiation, the characteristic cell markers at each stage, andimages of cells during iNK differentiation at day 0, day 6, day 21, andday 28.

FIG. 2B provides a schematic of the various cell stages during the iNKdifferentiation process and flow cytometry analysis of CD45, CD34, andCD43 expressing cells at Day 10 and flow cytometry analysis of CD7, CD45and CD38 expressing cells at Day 14.

FIG. 3A provides a graph demonstrating the percentages of cells withCD34⁺CD43⁻ and CD34⁺/CD43⁺ expression at days 6 and 10.

FIG. 3B provides a graph demonstrating the percentages of cells withCD45⁺/CD56⁻ expression and CD45⁺/CD56⁺ expression at days 10, 14, 20,28, and 35.

FIG. 3C provides flow cytometry analysis of SOX2 and OCT3/4 expressionin iPSC cells, cells at day 2 (“iNK d2”), day 4 (“iNK d4”), day 6 (“iNKd6”), and day 21 (“iNK d21”) of differentiation.

FIG. 3D shows Tru-seq analysis of KLRK1 (NKG2D), G2 MB, NCAM1 (CD56),CD34, KDR, and OCT3/4 expression during iNK differentiation.

FIG. 3E provides flow cytometry analysis of CD3 expression in cells atday 21 and day 35 of differentiation compared to positive controlT-cells.

FIG. 3F provides a graph demonstrating expression of differentiationmarkers in WT at Days 21, 28, and 37 of differentiation from iPSC to iNKcells. Cells were analyzed by flow cytometry for NKp44, NKp46, CD16,KIR2DL4, CD94, NKG2A, and KIR3DL2 expression.

FIG. 4 shows iNK expansion by the protocol described herein(feeder-free) and that one iPSC generated about 200-340 iNK in 28 days.

FIG. 5 provides flow cytometry analysis of Granzyme B and Perforinexpressing cells at Day 16 and Day 24 of differentiation and cells atDay 38 of differentiation co-incubated with K562 cells.

FIGS. 6A-6B provide graphs measuring K562 (FIG. 6A) and RPMI (FIG. 6B)cell killing by iNK cells. Differentiated iNK cells were cultured atdifferent E:T ratios with K562 or RPMI cells for 24 hours.

FIGS. 7A-7C provides graphs measuring Granzyme B (FIG. 7A), IFNγ (FIG.7B) and TNFα (FIG. 7C) levels in WT differentiated cells co-culturedwith RPMI cells at 1:1 ratio.

FIG. 8 shows a schematic of the Spin EB and modified Spin EB protocolsalong with the different components added at Day 0, Day 2, and Day 3.

FIG. 9 shows CD34 expression in cells differentiated with the componentslisted in FIG. 8.

FIG. 10 shows CD45 and CD56 expression in cells differentiated with thecomponents listed in FIG. 8.

FIG. 11 shows percent of CD34+ expression in cells after Day 6 ofdifferentiation cultured in APEL media or Stemflex media (SFM) with CHIRand/or Activin A (AA).

FIG. 12 shows expression of CD45⁺/CD56⁺, CD56⁺/NKG2D^(+*, CD)56⁺/CD94⁺,and CD56⁺/CD16⁺ cells in different mediums during Stage II ofdifferentiation (HSPCs to NK cells).

FIG. 13 shows expression of CD45 and CD56 in differentiating cellscultured with or without 50 μM β-mercaptoethanol (bME) on iNK cellinduction.

FIG. 14 shows DoE (Design of Experiment) I design. DoE experiments werecarried out from day 6 to day 20.

FIG. 15 shows DoE I medium formulation test and yield results.

FIG. 16 shows DoE II medium formulation test and yield results.

FIG. 17 shows day 20 flow cytometry analysis of CD45, CD56, and CD16expression of the DoE II experiments 17-19 from FIG. 16.

FIG. 18 shows a comparison of iNK cell cytotoxicity against K562 cellsgenerated in rotating spinner vessels versus static conditions andcompared to NK cells derived from peripheral blood (PB-NK).

FIGS. 19A-19C show cell density and medium change schedule modulate NKcell expansion rate. 0.75, 1, and 2 represent number of millions ofcells plated starting on Day 21, with no media change (FIG. 19A), onechange (FIG. 19B), or two media changes (FIG. 19C). Arrows indicate whenmedia was changed.

FIG. 20 shows NK cell activation assay (measuring CD16, CD56, andCD107a) with or without PMA (propidium monoazide)/Ionomycin.

FIG. 21 shows purity of NK cells differentiated from two differentsources of iPSC cells using AP.1.0, as measured at differentiation day20, 23, 26, and 28 using flow cytometry to detect CD56⁺CD45⁺ NK cells.

FIG. 22 shows DoE IV medium formulation test and yield results.

FIG. 23 shows DoE V medium formulation test and yield results.

FIG. 24A shows day 21 flow cytometry analysis of CD45 and CD56expression in differentiated cells after culturing in Stembrew orStemFlex media.

FIG. 24B shows percent of cells positive for the indicated cell markersat day 28 after differentiation in StemFlex or StemBrew media with orwithout nicotinamide (NAM).

FIG. 24C shows percent of cells positive for the indicated cell markersat day 35 after differentiation in StemFlex or StemBrew media with orwithout nicotinamide (NAM).

FIG. 25A shows K562 cell toxicity and FIG. 25B shows L428 cellcytotoxicity achieved in the presence of cells differentiated usingStemFlex or StemBrew media with or without nicotinamide.

FIG. 26A shows CD107a expression dynamics and FIG. 26B shows Perforinexpression dynamics upon PMA/ION stimulation of iNK derived from AP2.0.

FIG. 27 presents cytokine and granzyme secretion measurements uponPMA/ION stimulation of iNK derived from AP2.0.

FIG. 28 presents the percentage of iNK cells expressing the indicatedmarkers.

FIG. 29A shows a diagram of a cell differentiation process (usingAligned Process 2) in a bioreactor setting.

FIG. 29B shows levels of CD45⁺/CD56⁺ single cells or aggregate cellsdetected at different time points during the Aligned Process 2 inbioreactors.

FIG. 29C shows cytotoxic effect of bioreactor differentiated cells(differentiated using Aligned Process 2) against K562 cells.

FIG. 30 shows levels of CD45⁺CD56⁺ cells detected followingdifferentiation in different concentrations of CHIR-99021.

FIG. 31A shows blood marker expression and FIG. 31B shows myeloidprogenitor marker CD33 expression in differentiation day 20 culturesderived using AP2.0 with or without addition of WNT-C59 in NK-MED-006medium.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery of adifferentiation protocol for NK cells that provides a shorteneddifferentiation period relative to known differentiation protocols.Specifically, current differentiation protocols require 5-6 weeks togenerate NK cells, and typically utilize spin aggregation, adherentdifferentiation with feeder layers, and require cell sorting. As shownherein, a series of differentiation steps comprising various growthfactors, cytokines, and protein inhibitors and activators, contributesto a shortened differentiation protocol that does not require feedercells or cell sorting. Without wishing to be bound by theory, themethods described herein provide means to differentiate cells that ismore amenable to scale-up and/or manufacturing as the methods utilizecontrolled aggregation, do not require feeder layers or cell sorting,and has a shorter timeline, e.g., NK cells start developing at 14 daysand reach 70/6-90% within 3-4 weeks.

Further, the disclosure provides methods for differentiating stem and/orprogenitor cells comprising at least one gene-edit. Gene editing NKcells for therapeutic application is difficult due to their resistanceto gene delivery and editing. Without wishing to be bound by theory,differentiating a stem and/or progenitor cells comprising a gene-editallows for successful gene editing of NK cells by using thedifferentiation and gene editing methods described herein, such that thegene-edit is maintained in the differentiated cell.

Accordingly, the disclosure provides methods, compositions and kits fordifferentiating the cells described herein.

Methods of Differentiation

In some aspects, the disclosure provides methods and compositions fordifferentiating stem or progenitor cells into HSPCs and/or NK cells. Insome embodiments, HSPCs differentiated from stem or progenitor cellsusing the methods and compositions described herein are furtherdifferentiated into any cell in the hematopoietic lineage.

In some embodiments, stem or progenitor cells are differentiated into NKcells using any of the methods described herein. In some embodiments,stem or progenitor cells are differentiated into HSPCs using any of themethods described herein. In some embodiments, mesodermal cells aredifferentiated into NK cells. In some embodiments, hemogenic endotheliumis differentiated into NK cells. In some embodiments, HSPCs aredifferentiated into NK cells. In some embodiments, common lymphoidprogenitor cells are differentiated into NK progenitors. In someembodiments, common lymphoid progenitor cells are differentiated into NKcells. In some embodiments, NK progenitors are differentiated into NKcells. In some embodiments, common lymphoid progenitors or NKprogenitors are differentiated into innate lymphoid cells. In someembodiments, immature NK cells are differentiated into NK cells. In someembodiments, NK cells are further matured and differentiated to expressterminal and/or exhaustion markers. In some embodiments, inducedpluripotent stem cells (iPSCs) are differentiated into HSPCs. In someembodiments, iPSCs are differentiated into HSPCs which aredifferentiated into NK cells. It is noted that any of thedifferentiation methods provided herein may be performed in vitro or exvivo. Accordingly, in some embodiments, the methods for differentiatingNK cells or intermediary stem cells do not comprise a method fortreatment of the human or animal body by therapy. Likewise, in someembodiments, the methods for differentiating NK cells or intermediatestem cells (e.g., HSPCs) do not comprise methods for modifying the germline genetic identity of human beings.

Stage I: Differentiation of Stem Cells into HSPCs

In some embodiments, the disclosure provides compositions and methodsfor differentiating stem cells or progenitor cells into HSPCs.

In some embodiments, stem cells are differentiated into a cellpopulation comprising HSPCs using the following method:

(a) culturing a population of stem cells in a medium comprising anamount of a ROCK inhibitor under conditions sufficient to form apopulation comprising cell aggregates;

(b) culturing the population comprising cell aggregates in a mediumcomprising BMP-4;

(c) culturing the population comprising cell aggregates in a mediumcomprising BMP-4, FGF2, a WNT pathway activator, and Activin A;

(d) culturing the population comprising cell aggregates in a mediumcomprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and anactivin/nodal inhibitor to form a cell population comprisinghematopoietic stem and progenitor cells (HSPCs). In some embodiments,the medium of (b) comprises BMP-4 and a ROCK inhibitor.

In some embodiments, step (a) comprises culturing the population of stemcells for about 12-48 hours. In some embodiments, step (a) comprisesculturing the population of stem cells for about 12-24 hours. In someembodiments, step (a) comprises culturing the population of stem cellsfor about 16-20 hours. In some embodiments, step (b) comprises culturingthe population comprising cell aggregates for up to 24 hours. In someembodiments, step (b) comprises culturing the population comprising cellaggregates for about 4-24 hours. In some embodiments, step (b) comprisesculturing the population comprising cell aggregates for about 4-12hours. In some embodiments, step (b) comprises culturing the populationcomprising cell aggregates for about 6-10 hours. In some embodiments,step (c) comprises culturing the population comprising cell aggregatesfor about 1-3 days. In some embodiments, step (c) comprises culturingthe population comprising cell aggregates for about 2 days. In someembodiments, step (d) comprising culturing the population comprisingcell aggregates for about 1-3 days. In some embodiments, step (d)comprising culturing the population comprising cell aggregates for about2 days.

In some embodiments, step (a) comprises culturing the population of stemcells for about 12-48 hours; step (b) comprises culturing the populationcomprising cell aggregates for up to about 24 hours; step (c) comprisesculturing the population comprising cell aggregates for about 1-3 days;and step (d) comprises culturing the population comprising cellaggregates for about 1-3 days. In some embodiments, step (a) comprisesculturing the population of stem cells for about 16-20 hours; step (b)comprises culturing the population comprising cell aggregates for about6-10 hours; step (c) comprises culturing the population comprising cellaggregates for about 2 days; and step (d) comprises culturing thepopulation comprising cell aggregates for about 2 days.

In some embodiments, the time to generate aggregates in step (a) isabout 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36hours, about 42 hours, or about 48 hours. In some embodiments, the timeto generate aggregates in step (a) is about 16 hours, about 17 hours,about 18 hours, about 19 hours, or about 20 hours.

In some embodiments, differentiating a population of stem cells into acell population comprising HSPCs takes about 4-9 days. In someembodiments, differentiating a population of stem cells into a cellpopulation comprising HSPCs takes about 5-7 days.

In some embodiments, steps (a)-(d) form a cell population comprisingHSPCs that are then differentiated into NK cells using the methodsdescribed herein. In some embodiments, steps (a)-(d) form a cellpopulation comprising HSPCs that are then differentiated into any cellwithin the hematopoietic lineage using methods known to those of skillin the art.

Stem and Progenitor Cells

In some embodiments, stem or progenitor cells are differentiated intonatural killer (NK) cells. In some embodiments, stem or progenitor cellsare differentiated into HSPCs. In some embodiments, the stem orprogenitor cell is a mammalian cell. In some embodiments, the stem orprogenitor cell is a human cell. In some embodiments, the stem orprogenitor cell is a pluripotent stem cell (PSC). In some embodiments,the stem or progenitor cell is an embryonic stem cell (ESC), an adultstem cell (ASC), an induced pluripotent stem cell (iPSC), or ahematopoietic stem or progenitor cell (HSPC). In some embodiments, thestem or progenitor cell is an iPSC.

In some embodiments, the stem cells described herein (e.g., iPSCs) aregene-edited and then differentiated into a cell type of interest, e.g.,HSPC or NK cell. In some embodiments, the differentiated cell retainsthe gene-edits of the cell from which it is derived.

Stem cells are capable of both proliferation and giving rise to moreprogenitor cells, these in turn having the ability to generate a largenumber of mother cells that can in turn give rise to differentiated ordifferentiable daughter cells. The daughter cells themselves can beinduced to proliferate and produce progeny that subsequentlydifferentiate into one or more mature cell types, while also retainingone or more cells with parental developmental potential. The term “stemcell” refers then, to a cell with the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Insome embodiments, the term “progenitor” or “stem cell” refers to ageneralized mother cell whose descendants (progeny) specialize, often indifferent directions, by differentiation, e.g., by acquiring completelyindividual characteristics, as occurs in progressive diversification ofembryonic cells and tissues. Cellular differentiation is a complexprocess typically occurring through many cell divisions. Adifferentiated cell may derive from a multipotent cell that itself isderived from a multipotent cell, and so on. While each of thesemultipotent cells may be considered stem cells, the range of cell typesthat each can give rise to may vary considerably. Some differentiatedcells also have the capacity to give rise to cells of greaterdevelopmental potential. Such capacity may be natural or may be inducedartificially upon treatment with various factors. In many biologicalinstances, stem cells can also be “multipotent” because they can produceprogeny of more than one distinct cell type, but this is not requiredfor “stem-ness.”

A “differentiated cell” is a cell that has progressed further down thedevelopmental pathway than the cell to which it is being compared. Thus,stem cells can differentiate into lineage-restricted precursor cells(such as a hematopoietic stem and progenitor cell (HSPC)), which in turncan differentiate into other types of precursor cells further down thepathway (such as a common lymphoid progenitor cell), and then to anend-stage differentiated cell, such as a natural killer cell, whichplays a characteristic role in a certain tissue type, and may or may notretain the capacity to proliferate further.

Embryonic Stem Cells

In some embodiments, HSPCs and/or NK cells are differentiated fromembryonic stem cells (ESCs). ESCs are derived from blastocytes ofmammalian embryos and are able differentiate into any cell type andpropagate rapidly. ESCs are also believed to have a normal karyotype,maintaining high telomerase activity, and exhibiting remarkablelong-term proliferative potential, making these cells excellentcandidates for use as gene-edited stem cells. In some embodiments, HSPCsare differentiated from ESCs. In some embodiments, NK cells aredifferentiated from ESCs. In some embodiments, ESCs are differentiatedinto HSPCs and/or NK cells using any method described herein. In someembodiments, ESCs are gene-edited before differentiation into HSPCsand/or NK cells.

Adult Stem Cells

In some embodiments, HSPCs and/or NK cells are differentiated from adultstem cells (ASCs). ASCs are undifferentiated cells that may be found inmammals, e.g., humans. ASCs are defined by their ability to self-renew,e.g., be passaged through several rounds of cell replication whilemaintaining their undifferentiated state, and ability to differentiateinto several distinct cell types, e.g., glial cells. Adult stem cellsare a broad class of stem cells that may encompass hematopoietic stemcells, mammary stem cells, intestinal stem cells, mesenchymal stemcells, endothelial stem cells, neural stem cells, olfactory adult stemcells, neural crest stem cells, and testicular cells. In someembodiments, HSPCs are differentiated from ASCs. In some embodiments, NKcells are differentiated from ASCs. In some embodiments, ASCs aredifferentiated into HSPCs and/or NK cells using any method describedherein. In some embodiments, ASCs are gene-edited before differentiationinto HSPCs and/or NK cells.

Induced Pluripotent Stem Cells

In some embodiments, HSPCs and/or NK cells are differentiated frompluripotent stem cells (iPSCs). An iPSC may be generated directly froman adult human cell by introducing genes that encode criticaltranscription factors involved in pluripotency, e.g., Oct4, Sox2, cMyc,and Klf4. An iPSC may be derived from the same subject to whichsubsequent progenitor cells are to be administered. That is, a somaticcell can be obtained from a subject, reprogrammed to an inducedpluripotent stem cell, and then re-differentiated into a progenitor cellto be administered to the subject (e.g., autologous cells). However, inthe case of autologous cells, a risk of immune response and poorviability post-engraftment remain. In some embodiments, iPSC aregenerated from adult somatic cells using genetic reprogramming methodsknown in the art. In some embodiments, the iPSCs are derived from acommercial source. In some embodiments, HSPCs are differentiated fromiPSCs. In some embodiments, NK cells are differentiated from iPSCs. Insome embodiments, iPSCs are differentiated into HSPCs and/or NK cellsusing any method described herein. In some embodiments, iPSCs aregene-edited before differentiation into HSPCs and/or NK cells.

Mesoderm

In some embodiments, mesodermal cells are produced using thedifferentiation methods described herein. In some embodiments,mesodermal cells are an intermediate cell type between a stem cell andan HSPC. A mesodermal cell type is one of the three germinal layers inembryonic development. The mesoderm eventually differentiates in to, butis not limited to muscle, connective tissue, bone, red blood cells,white blood cells, and microglia. In some embodiments, mesodermal cellsare derived from any of the stem cells described herein. In someembodiments, mesodermal cells are derived from iPSC. In someembodiments, mesodermal cells have any of the gene-edits describedherein. In some embodiments, mesodermal cells are differentiated intoHSPCs. In some embodiments, mesodermal cells are differentiated into NKcells. In some embodiments, mesodermal cells are differentiated intoHSPCs and/or NK cells using any method described herein. In someembodiments, mesodermal cells are gene-edited before differentiationinto HSPCs and/or NK cells.

Hemogenic Endothelium

In some embodiments, hemogenic endothelium (HE) cells are produced usingthe differentiation methods described herein. In some embodiments, HEcells are an intermediate cell type between a stem cell and an HSPC.This cell type is an intermediate precursor of hematopoieticprogenitors. In some embodiments, HE cells are derived from any of thestem cells described herein. In some embodiments, HE cells are derivedfrom iPSC. In some embodiments, the HE cells have any of the gene-editsdescribed herein. In some embodiments, the HE cells are differentiatedinto HSPCs. In some embodiments, the HE cells are differentiated into NKcells. In some embodiments, HE cells are differentiated into HSPCsand/or NK cells using any method described herein. In some embodiments,HE cells are gene-edited before differentiation into HSPCs and/or NKcells.

Human Hematopoietic Stem and Progenitor Cells

In some embodiments, hematopoietic stem and progenitor cells (hHSPCs)are produced using the differentiation methods described herein. Thisstem cell lineage gives rise to all blood cell types, includingerythroid (erythrocytes or red blood cells (RBCs)), myeloid (monocytesand macrophages, neutrophils, basophils, eosinophils,megakaryocytes/platelets, and dendritic cells), and lymphoid (T-cells,B-cells, NK-cells). Blood cells are produced by the proliferation anddifferentiation of a very small population of pluripotent hematopoieticstem cells (HSCs) that also have the ability to replenish themselves byself-renewal. During differentiation, the progeny of HSCs progressthrough various intermediate maturational stages, generatingmulti-potential and lineage-committed progenitor cells prior to reachingmaturity. Bone marrow (BM) is the major site of hematopoiesis in humansand, under normal conditions, only small numbers of hematopoietic stemand progenitor cells (HSPCs) can be found in the peripheral blood (PB).Treatment with cytokines, some myelosuppressive drugs used in cancertreatment, and compounds that disrupt the interaction betweenhematopoietic and BM stromal cells can rapidly mobilize large numbers ofstem and progenitors into the circulation. In some embodiments, HSPCsare derived from any of the stem cells described herein. In someembodiments, HSPCs are derived from iPSCs. In some embodiments, theHSPCs have any of the gene-edits described herein. In some embodiments,the HSPCs cells are differentiated into NK cells. In some embodiments,HSPCs are differentiated into NK cells using any method describedherein. In some embodiments, HSPCs are gene-edited beforedifferentiation into NK cells.

Stage I Cell Phenotypes

In some embodiments, cell aggregates are maintained throughdifferentiation from stem cell to HSPC. In some embodiments, singlecells form during differentiation into HSPCs.

In some embodiments, the stem or progenitor cells are Oct3/4+ and Sox2+.In some embodiments, Oct3/4 and Sox2 expression is reduced as cellsdifferentiate into HSPCs. In some embodiments, the differentiating cellsare CD34⁺/CD43-. In some embodiments, CD43 expression increasesthroughout the differentiation process. In some embodiments, thepopulation of cells comprising HSPCs formed in step (d) of Stage Icomprises CD34⁺/CD43⁺/CD45⁻ cells. In some embodiments, the populationof cells comprising HSPCs formed in step (d) of Stage I compriseCD34⁺/CD43⁺/CD45⁺ cells. In some embodiments, the population of cellscomprising HSPCs formed in step (d) of Stage I comprise CD34⁺CD43-CD45⁺cells. In some embodiments, the cells in steps (a)-(d) are CD56-.

Stage II: Differentiation of HSPCs into NK Cells

In some embodiments, the disclosure provides compositions and methodsfor differentiating HSPCs into NK cells.

In some embodiments, a cell population comprising HSPCs isdifferentiated into a cell population comprising NK cells using thefollowing method:

(a) culturing the cell population in a medium comprising FGF2, VEGF,TPO, SCF, IL-3 and FLT3L;

(b) culturing the cell population in a medium comprising IL-3, IL-7,FLT3L, IL-15 and SCF;

(c) culturing the cell population in a medium comprising IL-7, FLT3L,IL-15 and SCF for a time sufficient to generate NK cells.

In some embodiments, step (a) comprises culturing the cell populationfor about 1-3 days. In some embodiments, step (a) comprises culturingthe cell population for about 2 days. In some embodiments, step (b)comprises culturing the cell population for up to 8 days. In someembodiments, step (b) comprises culturing the cell population for about6-8 days. In some embodiments, step (c) comprises culturing the cellpopulation for at least 6 days. In some embodiments, step (c) comprisesculturing the cell population for at least 6 days and up to 21-28 daystotal. In some embodiments, step (c) comprises culturing the cellpopulation for about 6-28 days. In some embodiments, step (a) comprisesculturing cell population for about 1-3 days; step (b) comprisesculturing the cell population for up to about 8 days; and step (c)comprises culturing the for at least 6 days. In some embodiments, step(a) comprises culturing the cell population for about 2 days; step (b)comprises culturing the cell population for about 6-8 days; and step (c)comprises culturing the cell population for about 6-28 days.

In some embodiments, differentiating a cell population comprising HSPCsinto a cell population comprising NK cells takes about 14-40 days. Insome embodiments, differentiating a cell population comprising HSPCsinto a cell population comprising NK cells takes about 14-17 days.

In some embodiments, a time sufficient to generate a first NK cell instep (c) is about 6 days, about 7 days, about 8 days, about 9 days,about 10 days, about 11 days, about 12 days, about 13 days, about 14days, or about 15 days. In some embodiments, a time sufficient togenerate a cell population comprising at least about 70% NK cells isabout 6 days, about 7 days, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 13 days, about 14 days, about 15days, about 16 days, about 17 days, about 18 days, about 19 days, about20 days, about 21 days, about 22 days, about 23 days, about 24 days,about 25 days, about 26 days, about 27 days, or about 28 days. In someembodiments, a time sufficient to generate a cell population comprisingat least about 80% NK cells is about 6 days, about 7 days, about 8 days,about 9 days, about 10 days, about 11 days, about 12 days, about 13days, about 14 days, about 15 days, about 16 days, about 17 days, about18 days, about 19 days, about 20 days, about 21 days, about 22 days,about 23 days, about 24 days, about 25 days, about 26 days, about 27days, or about 28 days. In some embodiments, a time sufficient togenerate a cell population comprising at least about 90% NK cells isabout 6 days, about 7 days, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 13 days, about 14 days, about 15days, about 16 days, about 17 days, about 18 days, about 19 days, about20 days, about 21 days, about 22 days, about 23 days, about 24 days,about 25 days, about 26 days, about 27 days, or about 28 days. In someembodiments, a time sufficient to generate a cell population comprisingat least about 95% NK cells is about 6 days, about 7 days, about 8 days,about 9 days, about 10 days, about 11 days, about 12 days, about 13days, about 14 days, about 15 days, about 16 days, about 17 days, about18 days, about 19 days, about 20 days, about 21 days, about 22 days,about 23 days, about 24 days, about 25 days, about 26 days, about 27days, or about 28 days. In some embodiments, a time sufficient togenerate a cell population comprising at least about 99% NK cells isabout 6 days, about 7 days, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 13 days, about 14 days, about 15days, about 16 days, about 17 days, about 18 days, about 19 days, about20 days, about 21 days, about 22 days, about 23 days, about 24 days,about 25 days, about 26 days, about 27 days, or about 28 days.

In some embodiments, a cell population comprising HSPCs isdifferentiated into a cell population comprising NK cells using thefollowing method:

(a) culturing the cell population in a medium comprising FGF2, VEGF,TPO, SCF, IL-3 and FLT3L;

(b) culturing the cell population in a medium comprising IL-3, IL-7,FLT3L, IL-15 and SCF;

(c) culturing the cell population in a medium comprising IL-7, FLT3L,IL-15 and SCF;

(d) culturing the cell population in a medium comprising IL-7, FLT3L,IL-15, SCF, and nicotinamide for a time sufficient to generate NK cells.

In some embodiments, a cell population comprising HSPCs isdifferentiated into a cell population comprising NK cells using thefollowing method:

(a) culturing the cell population in a medium comprising FGF2, VEGF,TPO, SCF, IL-3 and FLT3L;

(b) culturing the cell population in a medium comprising IL-3, IL-7,FLT3L, IL-15 and SCF;

(c) culturing the cell population in a medium comprising IL-7, FLT3L,IL-15 and SCF;

(d) culturing the cell population in a medium comprising IL-7, FLT3L,IL-15, and SCF for a time sufficient to generate NK cells.

In some embodiments, step (a) comprises culturing the cell populationfor about 1-3 days. In some embodiments, step (a) comprises culturingthe cell population for about 2 days. In some embodiments, step (b)comprises culturing the cell population for up to 8 days. In someembodiments, step (b) comprises culturing the cell population for about6-8 days. In some embodiments, step (c) comprises culturing the cellpopulation for up to 6 days. In some embodiments, step (c) comprisesculturing the cell population for about 6 days. In some embodiments,step (d) comprises culturing the cell population for at least 6 days andup to 10-16 days total. In some embodiments, step (d) comprisesculturing the cell population for about 8 to 16 days. In someembodiments, step (a) comprises culturing the cell population for about1-3 days; step (b) comprises culturing the cell population for up toabout 8 days; step (c) comprises culturing the cell population for up to6 days; and step (d) comprises culturing the cell population for atleast 6 days and up to 10-16 days total. In some embodiments, step (a)comprises culturing the cell population for about 2 days; step (b)comprises culturing the cell population for about 6-8 days; step (c)comprises culturing the cell population for about 6 days, and step (d)comprises culturing the cell population for about 8 to 16 days.

In some embodiments, differentiating a cell population comprising HSPCsinto a cell population comprising NK cells takes about 14-40 days. Insome embodiments, differentiating a cell population comprising HSPCsinto a cell population comprising NK cells takes about 14-19 days.

In some embodiments, a time sufficient to generate a first NK cell instep (d) is about 6 days, about 7 days, about 8 days, about 9 days,about 10 days, about 11 days, about 12 days, about 13 days, about 14days, about 15 days, or about 16 days. In some embodiments, a timesufficient to generate a cell population comprising at least about 70%NK cells in step (d) is about 6 days, about 7 days, about 8 days, about9 days, about 10 days, about 11 days, about 12 days, about 13 days,about 14 days, about 15 days, or about 16 days. In some embodiments, atime sufficient to generate a cell population comprising at least about80% NK cells in step (d) about 6 days, about 7 days, about 8 days, about9 days, about 10 days, about 11 days, about 12 days, about 13 days,about 14 days, about 15 days, or about 16 days. In some embodiments, atime sufficient to generate a cell population comprising at least about90% NK cells in step (d) is about 6 days, about 7 days, about 8 days,about 9 days, about 10 days, about 11 days, about 12 days, about 13days, about 14 days, about 15 days, or about 16 days. In someembodiments, a time sufficient to generate a cell population comprisingat least about 95% NK cells in step (d) is about 6 days, about 7 days,about 8 days, about 9 days, about 10 days, about 11 days, about 12 days,about 13 days, about 14 days, about 15 days, or about 16 days. In someembodiments, a time sufficient to generate a cell population comprisingat least about 99% NK cells in step (d) is about 6 days, about 7 days,about 8 days, about 9 days, about 10 days, about 11 days, about 12 days,about 13 days, about 14 days, about 15 days, or about 16 days.

Common Lymphoid Progenitor

In some embodiments, HSPCs are differentiated into CLPs. In someembodiments, CLPs are an intermediate cell type generated whiledifferentiating a stem or progenitor cell into NK cells. In someembodiments, CLPs are an intermediate cell type generated whiledifferentiating HSPCs to NK cells. CLPs are descendants of HSPCs. Thesecells differentiate into the lymphoid lineage of blood cells. Furtherdifferentiation yields B-cell progenitor cells, Natural Killer cells,and thymocytes. In some embodiments, CLP cells are derived from iPSCs.In some embodiments, CLP cells are differentiated from HSPCs. In someembodiments, the CLP cells have any of the gene-edits described herein.In some embodiments, the CLP cells are differentiated into NK cells. Insome embodiments, CLP cells are differentiated into NK cells using anymethod described herein. In some embodiments, CLP cells are gene-editedbefore differentiation into NK cells.

NK Progenitor Cells

In some embodiments, HSPCs are differentiated into NK progenitor cells(NKP) and immature NK cells. In some embodiments, CLPs differentiateinto a bipotent NK/T progenitor that can develop exclusively into Tand/or NK cells. In some embodiments, the transition from NK/Tprogenitor to NKP is marked by the acquisition of the IL-2/15Rb subunit(CD122 receptor). Expression of CD122 turns NKPs into IL-2/IL-15responsive cells that are committed to the NK cell lineage. NKP cellsare capable of differentiating into immature NK cells. In someembodiments, expression of growth factor receptors, such as FLT3 andIL-7Ra, decrease as cells proceed from NKP to immature NK cells, whereasthe expression of IL-2Rb, CD2 and 2B4 (CD244) increases. As describedherein, maturation of immature NK cells involves the acquisition ofactivation and inhibitory markers.

Innate Lymphoid Cells (ILCs)

In some embodiments, the methods described herein produce innatelymphoid cells (ILCs). ILCs are a growing family of immune cells thatmirror the phenotypes and functions of T cells. NK cells can beconsidered the innate counterparts of cytotoxic CD8⁺ T cells, whereasILC1s, ILC2s, and ILC3s may represent the innate counterparts of CD4⁺ Thelper 1 (TH1), TH2, and TH17 cells. However, in contrast to T cells,ILCs do not express antigen receptors or undergo clonal selection andexpansion when stimulated. Instead, ILCs react promptly to signals frominfected or injured tissues and produce an array of secreted cytokines,that direct the developing immune response into one that is adapted tothe original insult. ILCs develop from CLPs.

In some embodiments, NK cells are generally included in the ILC familybecause their phenotypic, developmental and functional propertiesoverlap considerably with those of ILCIs. In some embodiments, bothhuman ILCs and human NK cells express CD56 and NKp46. Accordingly, insome embodiments, ILCs are differentiated from stem cells using themethods described herein.

Stage II Cell Phenotypes

In some embodiments, the cell population comprises cell aggregates andsingle cells. In some embodiments, the cell aggregates dissociate intosingle cells. In some embodiments, the cell population comprises amajority of single cells.

In some embodiments, the HSPC cells are CD34⁺/CD43⁺/CD45⁻. In someembodiments, the HSPCs are differentiated to common lymphoidprogenitors. In some embodiments, the CLPs areCD34⁻/CD45⁺/CD38⁺/CD117⁺/CD7⁺. In some embodiments, CLPs are CD34+. Insome embodiments, CLPs are CD38⁻. In some embodiments, CLPs are CD117⁻.In some embodiments, CD56 expression increases in differentiating cells.In some embodiments, differentiating cells are CD45⁺/CD56⁺. In someembodiments, differentiating cells areCD34⁻/CD45⁺/CD56⁺/NKp46⁺/CD94⁺/NKG2A⁺. In some embodiments, NK cellsformed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16^(−/+)/KIR^(−/+). Insome embodiments, NK cells formed in step (c) or step (d) of Stage IIare CD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁺/KIR⁺. In someembodiments, NK cells formed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁻/KIR⁻. In someembodiments, NK cells formed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁺/KIR⁻. In someembodiments, NK cells formed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁻/KIR⁺. In someembodiments, the NK cells formed in step (c) or step (d) of Stage II donot express CD3.

In some embodiments, the NK cells formed in step (c) or step (d) areCD45⁺/CD56⁺.

In some embodiments, the NK cells formed in step (c) or step (d) ofStage II comprise at least one function of endogenous NK cells asdescribed herein.

Stage III: Expansion of NK Cells

In some embodiments, the disclosure provides compositions and methodsfor expanding differentiated NK cells.

In some embodiments, one iPSC generates about 200 to about 340 NK cellsduring differentiation. In some embodiments, one iPSC generates about200 to about 340 NK cells in 28 days. In some embodiments, one iPSCgenerates about 200 to about 340 NK cells in 34 days.

In some embodiments, NK cells are expanded without feeder cells. In someembodiments, NK cells are expanded with feeder cells. In someembodiments, culturing NK cells with K562 feeder cells enhances NK cellexpansion.

In some embodiments, NK cells are expanded in static cell cultureconditions. In some embodiments, NK cells are expanded in spinnercultures.

In some embodiments, NK cells are cultured for cell expansion in amedium comprising IL-15, IL-7, SCF, FLT3L, or any combination thereof.In some embodiments, NK cells are cultured for cell expansion in amedium comprising IL-15, IL-7, SCF and FLT3L. In some embodiments, NKcells are cultured for cell expansion in a medium comprising 15 ng/mLIL-15, 20 ng/mL IL-7, 20 ng/mL SCF, and 15 ng/mL FLT3L.

In some embodiments, fresh media is added two days after the start ofexpansion culture. In some embodiments, fresh media is added on thesecond day, and again on the third day after the start of expansionculture. In some embodiments, cells are cultured at 0.3×10⁶ cells/mL,0.5×10⁶ cells/mL, 1×10⁶ cells/mL, 1.1×10⁶ cells/mL, 1.2×10⁶ cells/mL,1.3×10⁶ cells/mL, 1.4×10⁶ cells/mL, 1.5×10⁶ cells/mL, 1.6×10⁶ cells/mL,1.7×10⁶ cells/mL, 1.8×10⁶ cells/mL, 1.9×10⁶ cells/mL, or 2.0×10⁶cells/mL for NK cell expansion. In some embodiments, cell density doesnot exceed 3.0×10⁶ cells/mL. In some embodiments, culture media isreplaced during expansion if the lactate concentration in the media isabove 13-16 mmol/L.

Cell Culture Conditions

In some embodiments, cells are washed in phosphate buffered salinebetween culture in different mediums. In some embodiments, cells arecultured in suspension culture. In some embodiments, cells are collectedby centrifugation after washing with PBS.

In some embodiments, cells are seeded at 3×10⁵, 4.0×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, or9×10⁷ cells per culture dish. One of skill in the art will know whatcell density is appropriate depending on the size of the culture dish.

In some embodiments the cells are cultured using a suspension agitationmethod. Methods for suspension agitation cell culture are known to thoseof skill in the art. In some embodiments, the suspension agitationcomprises rotation. In some embodiments the culture is under a 38rotation per minute (rpm) agitation condition. In some embodiments theculture is under a 39 rpm agitation condition. In some embodiments, theculture is under a 35 rpm agitation condition. In some embodiments, theculture is under a 45 rpm condition. In some embodiments the culture isunder a 50 rpm agitation condition. In some embodiments, the culture isunder a 65 rpm agitation condition. In some embodiments, the culture isunder a 75 rpm agitation condition. In some embodiments, the culture isunder a 98 rpm agitation. In some embodiments, the culture is under a110 rpm agitation condition.

In some embodiments, the culture is under an agitation condition betweenabout 30 and 115 rpm, between about 30 and 110 rpm, between about 30 and105 rpm, between about 30 and 100 rpm, between about 30 and 95 rpm,between about 30 and 90 rpm, between about 30 and 85 rpm, between about30 and 80 rpm, between about 30 and 75 rpm, between about 30 and 70 rpm,between about 30 and 65 rpm, between about 30 and 60 rpm, between about30 and 55 rpm, between about 30 and 50 rpm, between about 30 and 45 rpm,or between about 30 and 40 rpm. In some embodiments, the culture isunder an agitation condition between about 35 and 115 rpm, between about35 and 110 rpm, between about 35 and 105 rpm, between about 35 and 100rpm, between about 35 and 95 rpm, between about 35 and 90 rpm, betweenabout 35 and 85 rpm, between about 35 and 80 rpm, between about 35 and75 rpm, between about 35 and 70 rpm, between about 35 and 65 rpm,between about 35 and 60 rpm, between about 35 and 55 rpm, between about35 and 50 rpm, between about 35 and 45 rpm, or between about 35 and 40rpm.

In some embodiments, the culture is under an agitation condition betweenabout 40 and 115 rpm, between about 40 and 110 rpm, between about 40 and105 rpm, between about 40 and 100 rpm, between about 40 and 95 rpm,between about 40 and 90 rpm, between about 40 and 85 rpm, between about40 and 80 rpm, between about 40 and 75 rpm, between about 40 and 70 rpm,between about 40 and 65 rpm, between about 40 and 60 rpm, between about40 and 55 rpm, between about 40 and 50 rpm, or between about 40 and 45rpm. In some embodiments, the culture is under an agitation conditionbetween about 45 and 115 rpm, between about 45 and 110 rpm, betweenabout 45 and 105 rpm, between about 45 and 100 rpm, between about 45 and95 rpm, between about 45 and 90 rpm, between about 45 and 85 rpm,between about 45 and 80 rpm, between about 45 and 75 rpm, between about45 and 70 rpm, between about 45 and 65 rpm, between about 45 and 60 rpm,between about 45 and 55 rpm, or between about 45 and 50 rpm. In someembodiments, the culture is under an agitation condition between about50 and 115 rpm, between about 50 and 110 rpm, between about 50 and 105rpm, between about 50 and 100 rpm, between about 50 and 95 rpm, betweenabout 50 and 90 rpm, between about 50 and 85 rpm, between about 50 and80 rpm, between about 50 and 75 rpm, between about 50 and 70 rpm,between about 50 and 65 rpm, between about 50 and 60 rpm, or betweenabout 50 and 55 rpm. In some embodiments, the culture is under anagitation condition between about 55 and 115 rpm, between about 55 and110 rpm, between about 55 and 105 rpm, between about 55 and 100 rpm,between about 55 and 95 rpm, between about 55 and 90 rpm, between about55 and 85 rpm, between about 55 and 80 rpm, between about 55 and 75 rpm,between about 55 and 70 rpm, between about 55 and 65 rpm, or betweenabout 55 and 60 rpm.

In some embodiments, the culture is under an agitation condition betweenabout 60 and 115 rpm, between about 60 and 110 rpm, between about 60 and105 rpm, between about 60 and 100 rpm, between about 60 and 95 rpm,between about 60 and 90 rpm, between about 60 and 85 rpm, between about60 and 80 rpm, between about 60 and 75 rpm, between about 60 and 70 rpm,or between about 60 and 65 rpm. In some embodiments, the culture isunder an agitation condition between about 65 and 115 rpm, between about65 and 110 rpm, between about 65 and 105 rpm, between about 65 and 100rpm, between about 65 and 95 rpm, between about 65 and 90 rpm, betweenabout 65 and 85 rpm, between about 65 and 80 rpm, between about 65 and75 rpm, or between about 65 and 70 rpm. In some embodiments, the cultureis under an agitation condition between about 70 and 120 rpm, betweenabout 70 and 115 rpm, between about 70 and 110 rpm, between about 70 and105 rpm, between about 70 and 100 rpm, between about 70 and 95 rpm,between about 70 and 90 rpm, between about 70 and 85 rpm, between about70 and 80 rpm, or between about 70 and 75 rpm. In some embodiments, theculture is under an agitation condition between about 75 and 120 rpm,between about 75 and 115 rpm, between about 75 and 110 rpm, betweenabout 75 and 105 rpm, between about 75 and 100 rpm, between about 75 and95 rpm, between about 75 and 90 rpm, between about 75 and 85 rpm, orbetween about 75 and 80 rpm agitation condition.

In some embodiments, the culture is under an agitation condition betweenabout 80 and 120 rpm, between about 80 and 115 rpm, between about 80 and110 rpm, between about 80 and 105 rpm, between about 80 and 100 rpm,between about 80 and 95 rpm, between about 80 and 90 rpm, or betweenabout 80 and 85 rpm. In some embodiments, the culture is under anagitation condition between about 85 and 120 rpm, between about 85 and115 rpm, between about 85 and 110 rpm, between about 85 and 105 rpm,between about 85 and 100 rpm, between about 85 and 95 rpm, or betweenabout 85 and 90 rpm. In some embodiments, the culture is under anagitation condition between about 90 and 120 rpm, between about 90 and115 rpm, between about 90 and 110 rpm, between about 90 and 105 rpm,between about 90 and 100 rpm, or between about 90 and 95 rpm. In someembodiments, the culture is under an agitation condition between about95 and 120 rpm, between about 95 and 115 rpm, between about 95 and 110rpm, between about 95 and 105 rpm, or between about 95 and 100 rpm. Insome embodiments, the culture is under an agitation condition betweenabout 100 and 120 rpm, between about 100 and 115 rpm, between about 100and 110 rpm, or between about 100 and 105 rpm. In some embodiments, theculture is under an agitation condition between about 105 and 120 rpm,between about 105 and 115 rpm, or between about 105 and 110 rpm.

In various embodiments, the culture is under an agitation condition ofabout 30 to 40 rpm. For example, in some embodiments, the culture mayunder about 35 rpm agitation condition. In various embodiments, theculture is under an agitation condition of about 40 to 50 rpm. Forexample, the culture may be under about 45 rpm agitation condition. Invarious embodiments, the culture may be under an agitation conditionabout 60 and 70 rpm. For example, the culture may be about 65 rpmagitation condition.

In some embodiments, suspension agitation occurs in 6 well plates. Insome embodiments, suspension agitation occurs in roller bottles. In someembodiments, suspension agitation occurs in PBS spinner bottles.

Differentiation Medium Components

In some embodiments, the cells described herein are differentiated inmedia comprising growth factors and cytokines. In some embodiments, thecells are differentiated in media comprising inhibitors. In someembodiments, the cells are differentiated in media comprising one ormore of a ROCK inhibitor, GSK inhibitor, WNT pathway activator, WNTpathway inhibitor, Activin-A, activin/nodal inhibitor, TGFβ antagonist,FGF-2, VEGF, FLT3L, IL-3, IL-7, IL-15, BMP-4, SCF, TPO, WNT C-59, IL-2,IL-12, IL-21, IL-18, IL-27, IL-33, TGFβ1, aryl hydrocarbon antagonistand their enhancers: StemRegenin-1, UM729.

In some embodiments, the cells are cultured in a medium comprising atleast StemFlex medium. In some embodiments, the cells are cultured in amedium comprising at least StemBrew Basal Media. In some embodiments,the cells are cultured in a medium comprising a StemBrew Supplement. Insome embodiments, the cells are cultured in a medium comprising at leastalbumin polyvinylalcohol essential lipids (APEL) medium. In someembodiments, the cells are cultured in a medium comprising at leastSTEMdiff™ APEL™ medium. In some embodiments, the cells are cultured in amedium comprising at least STEMdiff™ APEL™ 2 medium. In someembodiments, the term “APEL medium” refers to STEMdiff™ APEL™ medium orSTEMdiff™ APEL™ 2 medium. In some embodiments, the cells are cultured ina medium comprising at least DMEM/F12 medium. In some embodiments, thecells are cultured in a medium comprising at least DMEM (highglucose)/F12 medium. In some embodiments, the cells are cultured in amedium comprising at least RPMI medium. In some embodiments, the cellsare cultured in a medium comprising at least MCDB131 medium. In someembodiments, the cells are cultured in a medium comprising at least IMDMmedium. In some embodiments, the cells are cultured in a mediacomprising at least the same, or similar components to any one ofStemFlex, StemBrew, STEMdiff APEL medium, STEMdiff, APEL 2 medium, DMEM,or DMEM/F12. In some embodiments, the cells are cultured in a mediacomprising at least the same, or similar components to DMEM (highglucose) or DMEM (high glucose)/F12. In some embodiments, the mediacomprises one or more of human serum, zinc sulfate, ethanolamine,β-mercaptoethanol, glucose, nicotinamide, or glutamax (e.g., glutaminesubstitute). In some embodiments, the media comprises DMEM/F12 and oneor more of human serum, zinc sulfate, ethanolamine, β-mercaptoethanol,glucose, nicotinamide, or glutamax. In some embodiments, the mediacomprises DMEM (high glucose)/F12 and one or more of human serum, zincsulfate, ethanolamine, β-mercaptoethanol, glucose, nicotinamide, orglutamax.

In some embodiments, the cells are cultured in a medium described hereinfor one hour to 28 days. In some embodiments, the cells are cultured ina medium described herein for 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

GSK Inhibitor and WNT Pathway Activators

As used herein, the term “GSK-3 inhibitor” refers to a compound or agroup of compounds, capable of inhibiting glycogen synthase kinase 3(GSK-3; either fully or partially). Glycogen synthase kinase 3 is aserine/threonine protein kinase that mediates the addition of phosphatemolecules onto serine and threonine amino acid residues. Phosphorylationof a protein by GSK-3 usually inhibits the activity of its downstreamtarget. GSK-3 has been shown to be integrally tied to pathways of cellproliferation and apoptosis. For example, GSK-3 has been shown tophosphorylate beta-catenin, resulting in beta-catenin being targeted fordegradation. GSK-3 is therefore a part of the canonical beta-catenin/Wntpathway, which signals the cell to divide and proliferate. GSK-3 alsoparticipates in several apoptotic signalling pathways by phosphorylatingtranscription factors that regulate apoptosis. GSK-3 can promoteapoptosis by both activating pro-apoptotic factors, such as p53, forexample, and inactivating survival-promoting factors throughphosphorylation.

In some embodiments, the GSK-3 inhibitor is, but is not limited to,valproic acid sodium salt, staurosporine, KT 5720 (CAS 108068-98-0),GSK-3 Inhibitor IX (CAS 667463-62-9), Ro 31-8220 (CAS 138489-18-6),SB-216763 (CAS 280744-09-4), CID 755673 (CAS 521937-07-5), Kenpaullone(CAS 142273-20-9), lithium chloride, GSK-3beta Inhibitor XII (TWS119;CAS 601514-19-6), GSK-3 Inhibitor XVI (CAS252917-06-9),lOZ-Hymenialdisine (CAS 82005-12-7), Indirubin (CAS 479-41-4),CHIR-98014 (CAS 252935-94-7), GSK-3beta Inhibitor VI (CAS 62673-69-2),Manzamine A (CAS 104196-68-1), Indirubin-3prime-monoxime (CAS160807-49-8), GSK-3 Inhibitor X (CAS 740841-15-0), GSK-3 Inhibitor XV,SB-415286 (CAS 264218-23-7), 1—Azakenpaullone (CAS 676596-65-9), TWS 119ditrifluoroacetate (CAS 601514-19-6), 5-Iodo-indirubin-3′-monoxime,GSK-3beta Inhibitor I (CAS 327036-89-5), 9-Cyanopaullone,Indirubin-5-sulfonic acid sodium salt, GSK-3beta inhibitor VII (CAS99-73-0), Cdk1/5 inhibitor (CAS 40254-90-8), Hymenidin (CAS107019-95-4), bisindolylmaleimide X hydrochloride (CAS 131848-97-0), 3F8(CAS 159109-11-2), isogranulatimide (CAS 244148-46-7), CR8, (R)-isomer(CAS 294646-77-8) L-779,450 (CAS 303727-31-3),indirubin-3prime-monoxime-5-sulphonic acid (CAS 331467-05-1), GSK-3Inhibitor II (CAS 478482-75-6), GSK-3beta Inhibitor VIII (CAS487021-52-3), Aloisine A (CAS 496864-16-5), GSK-3beta Inhibitor XI (CAS626604-39-5), GSK-3 Inhibitor IX (CAS 710323-61-8), Alsterpaullone,2-Cyanoethyl (CAS 852529-97-0), TCS 2002 (CAS 1005201-24-0), TCS 21311(CAS 1260181-14-3), A 1070722 (CAS 1384424-80-9), Ro-31-8220 (CAS138489-18-6), Enzastaurin (CAS 138489-18-6), MeBIO (CAS 667463-95-8),Cdk2/9 Inhibitor (CAS 507487-89-0), Cdk1/2 Inhibitor III (CAS443798-55-8), PHA 767491 hydrochloride (CAS 845714-00-3), AR-AO14418-d3, Indole-3-acetamide (CAS 879-37-8), Hymenialdisine Analogue 1(CAS 693222-51-4), CHIR-99021 (also known as6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino] ethyl] amino]-3-pyridinecarbonitrile and CT99021; CAS252917-06-9), CHIR-98014 (CAS 556813-39-9),(2′Z,3′E)-6-Bromoindirubin-3′-oxime (Bio; CAS 667463-62-9),Bio-Acetoxime (CAS 667463-85-6), SB216763 (CAS 280744-09-4), andcombinations thereof.

In some embodiments, the GSK-3 inhibitor is, but is not limited to,CHIR-99021, (2′Z,3′E)-6-Bromoindirubin-3′-oxime (Bio; CAS 667463-62-9),Kenpaullone (CAS 142273-20-9), GSK-3beta Inhibitor XII (TWS 119; CAS601514-19-6), Bio-Acetoxime (CAS 667463-85-6), CHIR-98014, SB216763 (CAS280744-09-4), GSK-3beta Inhibitor VIII (CAS 487021-52-3), andcombinations thereof. In some embodiments, the GSK-3 inhibitor isCHIR-99021 or a derivative thereof.

In some embodiments, the GSK-3 inhibitor is present in a concentrationof between 0.001 μM to 15 μM. In some embodiments, the GSK3-inhibitor isabout 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM,about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12μM, about 13 μM, about 14 μM, or about 15 μM. In some embodiments, theGSK-3 inhibitor is about 6 μM. In some embodiments, the GSK-3 inhibitoris about 7 μM.

As used herein, a “WNT pathway activator” or “a WNT agonist” is amolecule that mimics or increases WNT signaling. A WNT agonist is not tobe restricted to a molecule acting directly on WNT as the molecule mayact elsewhere in the WNT signaling pathway.

Non-limiting examples of WNT agonists include small molecules CHIR-99021(CAS 252917-06-9), a 2-amino-4,6-disubstituted pyrimidine, e.g. BML 284(CAS 853220-52-7), SKL 2001 (CAS 909089-13-0), WAY 262611 (CAS1123231-07-1), WAY 316606 (CAS 915759-45-4), SB 216763 (CAS280744-09-4), IQ 1 (CAS 331001-62-8), QS 11 (CAS 944328-88-5),deoxycholic acid (CAS 83-44-3), BIO (CAS 667463-62-9), kenpaullone (CAS142273-20-9), or a (hetero) arylpyrimidine. In some embodiments, a WNTagonist is an agonist antibody or functional fragment thereof or anantibody-like polypeptide.

In some embodiments, the WNT agonist is CHIR-99021 ((CHIR) CAS252917-06-9). In some embodiments, the medium comprises about 2 μM,about 2.5 μM, about 3 μM, about 3.5 μM, about 4.5 μM, about 5 μM, about5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM,about 8.5 μM, or about 9 μM CHIR-99021. In some embodiments, the mediumcomprises about 6 μM CHIR-99021. In some embodiments, the mediumcomprises about 7 μM CHIR-99021.

In some embodiments, the Wnt pathway activator is, but is not limitedto, IQ-1 and Wnt3a.

In some embodiments, the Wnt pathway activator, is present in aconcentration of between 1 ng/mL to 150 ng/mL, between 10 ng/mL to 100ng/mL, between 1 ng/mL to 50 ng/mL, between 45 ng/mL to 75 ng/mL,between 60 ng/mL to 110 ng/mL, between 115 ng/mL to 150 ng/mL, about 10ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL,about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL,about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, about100 ng/mL, about 105 ng/mL, about 110 ng/mL, about 115 ng/mL, about 120ng/mL, about 125 ng/mL, about 130 ng/mL, about 140 ng/mL, about 145ng/mL, or about 150 ng/mL.

Rock Inhibitors

Rho associated kinases (ROCK) are serine/threonine kinases that servedownstream effectors of Rho kinases (of which three isoforms exist—RhoA,RhoB and RhoC). ROCK inhibitors suitable for use in compositionscontemplated herein include, but are not limited to, polynucleotides,polypeptides, and small molecules. ROCK inhibitors contemplated hereinmay decrease ROCK expression and/or ROCK activity. Illustrative examplesof ROCK inhibitors contemplated herein include, but are not limited to,anti-ROCK antibodies, dominant negative ROCK variants, siRNA, shRNA,miRNA and antisense nucleic acids that target ROCK.

In some embodiments, the ROCK inhibitors include, but are not limitedto: thiazovivin, Y27632, Fasudil, AR122-86, RevitaCell™ Supplement,H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A,SB-772077-B, N-(4-Pyridyl)-N′-(2,4,6-trichlorophenyl)urea,3-(4-Pyridyl)-1H-indole, and(R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide,H-100, and ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, whichis herein incorporated by reference in its entirety.

In some embodiments, the ROCK inhibitor is thiazovivin, Y27632, orpyrintegrin. In some embodiments, the ROCK inhibitor is thiazovivin. Insome embodiments, the ROCK inhibitor is Y27632.

In some embodiments, the ROCK inhibitor is present at a concentration ofabout 1-15 pM, 5-15 μM, 1-30 μM, 5-30 μM, or about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 μM, or any range derivable therein. In some embodiments, theROCK inhibitor is present at a concentration of about 10 μM.

Activin/Nodal Inhibitor

As used herein, an “activin/nodal inhibitor” is a molecule that inhibitsor decreases activin signaling. An activin/nodal is not to be restrictedto a molecule acting directly on activin as the molecule may actelsewhere in the activin signaling pathway.

In some embodiments, activin/nodal inhibitors include small molecules SB431542 (CAS 301836-41-9), SB 505124 (CAS 694433-59-5), LDN 193189 (CAS1062368-24-4), LDN 193719 (CAS 1062368-49-3), Dorsomorphin (CAS866405-64-3), A 83-01 (CAS 909910-43-6), DMH 1 (CAS 1206711-16-1),RepSox (CAS 446859-33-2), or LY 364947 (CAS 396129-53-6). In someembodiments, the activin/nodal inhibitor is SB 431542.

In some embodiments, an activin/nodal inhibitor is an anti-activinantagonist antibody or functional fragment thereof or an antibody-likepolypeptide. In some embodiments, the activin/nodal inhibitor isFollistatin.

In some embodiments, the medium comprises 2 μM, about 2.5 μM, about 3μM, about 3.5 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM,about 6.5 μM, or about 7 μM of activin/nodal inhibitor. In someembodiments, the medium comprises about 2 μM, about 2.5 μM, about 3 μM,about 3.5 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about6.5 μM, or about 7 μM SB 431542. In some embodiments, the mediumcomprises about 5 μM SB 431542.

Porcn Inhibitor

In some embodiments, any media described herein comprises a PorcnInhibitor. Porcupine (Porcn) is a membrane-bound-O-acyltransferase.Porcn affects Wnt signaling by palmitoleating the Wnts and is essentialfor Wnt secretion and function. In some embodiments, the Porcn inhibitoris selected from LGK974 (Liu et al., Proc Natl Acad Sci USA. 2013December 10; 110(50):20224-9; Jiang et al., ProcNatl Acad Sci USA. 2013Jul. 30; 110(31):12649-54); Wnt C-59 (Proffitt et al., Cancer Res. 2013Jan. 15; 73(2):502-7); ETC-159 and ETC-131 (aka ETC-1922159, Madan etal., Oncogene. 2016 Apr. 28; 35(17): 2197-2207); IWP compounds includingIWP-L6 (Chen et al., Nat Chem Biol. 2009 February; 5(2): 100-7; Wang etal., J Med Chem. 2013 Mar. 28; 56(6):2700-4; Dodge et al., J Biol Chem.2012 Jun. 29; 287(27):23246-54); GNF6231 (Liu et al., Annals of theRheumatic Diseases Published Online First: 2 Feb. 2017, doi:10.1136/annrheumdis-2016-210294); Compounds 3-5 (Duraiswamy et al., JMed Chem. 2015 Aug. 13; 58(15):5889-99); Compound 6 (Poulsen, et al., J.Chem. Inf. Model., 55 (2015), p. 1435) and other porcupine inhibitors.In some embodiments, the Porcn inhibitor is Wnt C-59.

In some embodiment, the Porcn inhibitor is included in any mediumdescribed herein at a concentration of about 0.5-5 μM, about 1-15 μM,about 5-15 μM, about 10-20 μM, about 1-20 μM, or about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM. In someembodiments, the porcn inhibitor is present in the medium at aconcentration of about 2 μM. In some embodiments, Wnt C-59 is includedin any medium described herein at a concentration of about 0.5-5 μM,1-15 μM, 5-15 μM, 10-20 μM, 1-20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM. In some embodiments,Wnt C-59 is present in the medium at a concentration of about 2 μM.

FGF

In some embodiments, any medium described herein comprises fibroblastgrowth factor. Basic fibroblast growth factor, also referred to as bFGFor FGF-2, is a growth factor which has been implicated in diversebiological processes, including limb and nervous system development,wound healing, and tumor growth. Previous studies have indicated thatbFGF is unlikely to affect hematopoietic cell development or survival(Ratajczak et al., 1996), although bFGF has been used to supportfeeder-independent growth of human embryonic stem cells (Ludwig et al.,(2006).

In some embodiments, the bFGF is FGF2. In some embodiments, the FGF2 isa 146 amino acid FGF2 polypeptide (see e.g., R&D Systems Cat#AFL233-025). In some embodiments, the FGF2 is a 154 amino acid FGF2polypeptide (see e.g., Cell Guidance Systems Cat #GFH146-10).

In some embodiments, other fibroblast growth factors such as acidic FGF(aFGF), FGF4, FGF8, FGF9, FGF17 or FGF18 may substituted for or includedwith bFGF, e.g., at the concentrations described above. Alternately, anFGF-2 mimicking compounds may be substituted for FGF-2 to producesubstantially or essentially the same effect. FGF-2 mimics include FGF-2mimicking peptides, antibodies, and small molecules. For example,synthetic peptide F2A4-K-NS mimics the effects of FGF-2 in vitro and invivo (Lin et al., 2006) and may be substituted for FGF-2 in variousembodiments of the medium. FG loop (FGL) peptide is another example of aFGF-2 mimetic which is used in some embodiments of the medium. FGL is a15 amino acid sequence in the second F3 module of NCAM that represents apart of the binding site of NCAM to the FGFR1. FGL has been shown tobind to and activate FGFR1 and to stimulate neurite outgrowth (Kiselyovet al., 2003).

In some embodiments, the BioSET F2A peptide may also be substituted forFGF-2. The BioSET F2A peptide is a synthetic mimetic of the naturalhuman FGF-2 growth factor. The BioSET F2A peptide and the F2A4-KNSpeptide are available from FYI Tornier, Inc., or BioSurface EngineeringTechnologies, Inc. (“BioSET”). It is envisioned that combinations ofFGF-2 mimicking compounds may also be substituted for FGF-2 in variousembodiments of the medium.

In some embodiments, FGF is a mammalian FGF. In some embodiments, FGF ismouse FGF. In some embodiments, FGF is human FGF. In some embodiments,FGF is recombinant human FGF. In some embodiments, FGF-2 is a mammalianFGF-2. In some embodiments, FGF-2 is human FGF-2. In some embodiments,FGF-2 is recombinant human FGF-2.

In some embodiments, bFGF is included in any medium described herein ata concentration of from about 5 to about 100 ng/mL, 5 to about 50 ng/mL,from about 5 to about 25 ng/mL, or any range derivable therein. In someembodiments, bFGF is at a concentration of about 2.5, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or about110 ng/mL. In some embodiments, FGF is at a concentration of about 20ng/mL. In some embodiments, FGF is at a concentration of about 100ng/mL.

Bone Morphogenic Protein

In some embodiments, any media described herein comprises a bonemorphogenic protein (BMP) activator. In some embodiments, the mediacomprises BMP-4. Bone morphogenetic protein-4 (BMP-4) is a member of thegroup of bone morphogenic proteins and a ventral mesoderm inducer. BMPsare expressed in adult human bone marrow (BM) and are important for boneremodeling and growth. In some embodiments, inclusion of BMP4 is onlyneeded for the first two to three days in culture, after which time itcan be removed from the system with no detrimental effect ondifferentiation.

In some embodiments, the BMP is BMP2, BMP6, or BMP7.

BMP-4 is important for the modulation of the proliferative anddifferentiative potential of hematopoietic progenitor cells (Bhardwaj etal., 2001; Bhatia et al., 1999; Chadwick 2003). Additionally, BMP-4 canmodulate early hematopoietic cell development in human fetal, neonatal,and adult hematopoietic progenitor cells (Davidson and Zon, 2000; Huberet al., 1998; Marshall et al., 2000). For example, BMP-4 can regulatethe proliferation and differentiation of highly purified primitive humanhematopoietic cells from adult and neonatal sources (Bhatia et al.,1999), and BMP-4 can promote hematopoietic differentiation in humanembryonic stem cells (Chadwick, 2003).

In some embodiments, BMP-4 is a mammalian BMP-4. In some embodiments,BMP-4 is mouse BMP-4. In some embodiments, BMP-4 is human BMP-4. In someembodiments, BMP-4 is recombinant human BMP-4.

In some embodiments, BMP-4 is present in the medium at a concentrationof about 5-100 ng/mL, about 20-100 ng/mL, about 20-50 ng/mL, about 10-30ng/mL, about 15-30 ng/mL, about 20-30 ng/mL, or any range derivabletherein. In some embodiments, BMP-4 is included in the medium at aconcentration of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50ng/mL. In some embodiments, BMP-4 is included in the medium at aconcentration of about 30 ng/mL.

FLT3L

In some embodiments, any media described herein comprises Flt3 ligand(FLT3L). Flt3 ligand, also referred to as FLT-3 ligand, is theendogenous ligand for FLT3. FLT3 is a receptor tyrosine kinase expressedby immature hematopoietic progenitor cells. The ligand for FLT3 is atransmembrane or soluble protein and is expressed by a variety of cellsincluding hematopoietic and marrow stromal cells; in combination withother growth factors, Flt3 ligand can stimulate the proliferation anddevelopment of stem cells, myeloid and lymphoid progenitor cells,dendritic cells and natural killer cells. Activation of the receptorleads to tyrosine phosphorylation of various key adaptor proteins knownto be involved in different signal transduction pathways that controlproliferation, survival and other processes in hematopoietic cells. FLT3and mutations affecting FLT3 are also important in pathologicaldiseases, such as the prognosis and therapy of leukemia (Drexler et al.,2004).

In some embodiments, FLT3L is a mammalian FLT3L. In some embodiments,FLT3L is mouse FLT3L. In some embodiments, FLT3L is human FLT3L. In someembodiments, FLT3L is recombinant human FLT3L.

In some embodiments, Flt3 ligand is included in a culture medium at aconcentration of from 5 to about 100 ng/mL, 5 to about 50 ng/mL, fromabout 10 to about 20 ng/mL, from about 10 to about 30 ng/mL, from about15 to about 30 ng/mL, from about 20 to about 30 ng/mL, or any rangederivable therein. In some embodiments, Flt3 ligand is included in themedium at a concentration of about 2.5, 5, 10, 15, 20, 25, 30, 35, 40,45, or about 50 ng/mL.

In some embodiments, the concentration of FLT3L in the medium is about 1ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL,about 60 ng/mL, or about 70 ng/mL. In some embodiments, theconcentration of FLT3L in the medium is about 15 ng/mL or about 20ng/mL.

TPO

In some embodiments, any medium described herein comprisesthrombopoietin (TPO). TPO is a glycoprotein hormone which is primarilyproduced in vivo by the liver and kidney and is involved in the in vivogeneration of platelets in the bone marrow.

In some embodiments, TPO is a mammalian TPO. In some embodiments, TPO ismouse TPO. In some embodiments, TPO is human TPO. In some embodiments,TPO is recombinant human TPO.

In some embodiments, TPO is included in the medium at a concentration offrom about 2.5 to about 100 ng/mL, 5 to about 75 ng/mL, from about 10 toabout 50 ng/mL, from about 15 to about 35 ng/mL, at about 25 ng/ml, orany range derivable therein. In some embodiments, TPO is included in thedefined culture media at a concentration of about 2.5, 5, 10, 15, 20,25, 30, 35, 40, 45 or about 50 ng/mL. In some embodiments, theconcentration of TPO in the medium is about 10 ng/mL, about 15 ng/mL,about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40ng/mL, about 45 ng/mL, or about 50 ng/mL. In some embodiments, theconcentration of TPO in the medium is about 20 ng/mL.

IL-3

In some embodiments, any medium described herein comprises IL-3.Interleukin-3 (IL-3) is a hematopoietic growth factor involved in thesurvival, proliferation and differentiation of multipotent hematopoieticcells.

In some embodiments, IL-3 is a mammalian IL-3. In some embodiments, IL-3is mouse IL-3. In some embodiments, IL-3 is human IL-3. In someembodiments, IL-3 is recombinant human IL-3.

In some embodiments, IL-3 is included in the medium at a concentrationof from 2.5 to about 50 ng/mL, 2.5 to about 50 ng/mL, from about 5 toabout 50 ng/mL, from about 5 to about 25 ng/mL, from about 5 to about 15ng/mL, or any range derivable therein. In some embodiments, IL-3 isincluded in the medium at a concentration of about 2.5, 5, 10, 15, 20,25, or about 30 ng/mL. In some embodiments, the concentration of IL-3 inthe medium is about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL,about 45 ng/mL, or about 50 ng/mL.

In some embodiment, the IL-3 concentration is 5 ng/mL. In someembodiments, the IL-3 concentration is 40 ng/mL.

VEGF

In some embodiments, any medium described herein comprises VEGF.Vascular endothelial growth factor (VEGF) is an important signalingprotein which is involved in formation of the embryonic circulatorysystem and angiogenesis. VEGF can affect a variety of cell typesincluding vascular endothelium and other cell types (e.g., neurons,cancer cells, kidney epithelial cells). In vitro, VEGF can stimulateendothelial cell mitogenesis and cell migration. VEGF function has alsobeen shown to be important in a variety of disease states includingcancer, diabetes, autoimmune diseases, and ocular vascular diseases.

In some embodiments, VEGF is a mammalian VEGF. In some embodiments, VEGFis mouse VEGF. In some embodiments, VEGF is human VEGF. In someembodiments, VEGF is recombinant human VEGF.

In some embodiments, VEGF is included in the medium at a concentrationof from about 10-100 ng/mL, about 20-100 ng/mL, about 10-50 ng/mL, about15-30 ng/mL, about 20-30 ng/mL, about 20-50 ng/mL, or any rangederivable therein. In some embodiments, VEGF is included in the definedculture media at a concentration of about 2.5, 5, 10, 15, 20, 25, 30,35, 40, 45, or about 50 ng/mL. In some embodiments, the VEGFconcentration is 20 ng/mL.

IL-15

In some embodiments, any medium described herein comprises IL-15.Interleukin-15 (IL-15) is a cytokine that induces proliferation ofnatural killer cells.

In some embodiments, IL-15 is a mammalian IL-15. In some embodiments,IL-15 is mouse IL-15. In some embodiments, IL-15 is human IL-15. In someembodiments, IL-15 is recombinant human IL-15.

In some embodiments, the concentration of IL-15 in the medium is about 1ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL,about 60 ng/mL, or about 70 ng/mL.

In some embodiments, the concentration of IL-15 in the medium is about 1ng/ml to about 10 ng/mL, about 5 ng/mL to about 15 ng/mL, about 10 ng/mLto about 20 ng/mL, or about 15 ng/mL to about 25 ng/mL. In someembodiments, the concentration of IL-15 in the medium is about 15 ng/mL.

IL-7

In some embodiments, the medium comprises IL-7. Interleukin-7 (IL-7) isa hematopoietic growth factor that stimulates the differentiation ofhematopoietic stem cells.

In some embodiments, IL-7 is a mammalian IL-7. In some embodiments, IL-7is mouse IL-7. In some embodiments, IL-7 is human IL-7. In someembodiments, IL-7 is recombinant human IL-7.

In some embodiments, the IL-7 concentration in the medium is about 1ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL,about 60 ng/mL, or about 70 ng/mL. In some embodiments, the IL-7concentration in the medium is about 20 ng/mL.

SCF

In some embodiments, the medium comprises one or more hematopoieticcytokines. In some embodiments, the hematopoietic cytokine is stem cellfactor (SCF). Stem cell factor is a cytokine which binds CD117 (c-Kit).SCF is also known as “KIT ligand,” “c-kit ligand,” or “steel factor.”SCF exists in two forms: cell surface bound SCF and soluble (or free)SCF. Soluble SCF is typically produced in vivo by the cleavage ofsurface bound SCF by metalloproteases. SCF can be important for thesurvival, proliferation, and differentiation of hematopoietic stem cellsand other hematopoietic progenitor cells. In vivo, SCF can change theBFU-E (burst-forming unit-erythroid) cells, which are the earliesterythrocyte precursors in the erythrocytic series, into the CFU-E(colony-forming unit-erythroid).

In some embodiments, SCF is a mammalian SCF. In some embodiments, SCF ismouse SCF. In some embodiments, SCF is human SCF. In some embodiments,SCF is recombinant human SCF.

In some embodiments, SCF is included in the medium at a concentration offrom about 5 to about 100 ng/mL, 5 to about 50 ng/mL, from about 10 toabout 30 ng/mL, from about 15 to about 30 ng/mL, from about 20 to about30 ng/mL, or any range derivable therein. In some embodiments, SCF isincluded in the medium at a concentration of about 2.5, 5, 10, 15, 20,25, 30, 35, 40, 45, or about 50 ng/mL. In some embodiments, theconcentration of SCF in the medium is about 1 ng/mL, about 10 ng/mL,about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL,about 110 ng/mL, about 120 ng/mL, about 150 ng/mL, or about 200 ng/mL.In some embodiments, the concentration of SCF in the medium is about 100ng/mL. In some embodiments, the concentration of SCF in the medium isabout 20 ng/mL.

Nicotinamide

In some embodiments, the medium comprises nicotinamide. Nicotinamide isa form of vitamin B3 and aids in the differentiation of NK cells.

In some embodiments, the concentration of nicotinamide in the medium isabout 1 mM to 15 mM, from about 2 mM to 10 mM, from about 4 mM to 8 mM,or from about 5 mM to 7 mM. In some embodiments, the concentration ofnicotinamide in the medium is about 2 to 8 mM, from about 3 to 8 mM,from about 4 to 8 mM, from about 5 to 8 mM or from about 6 to 7 mM. Insome embodiments, the concentration of nicotinamide in the medium isabout 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM,about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, or about15 mM. In some embodiments, the concentration of nicotinamide in themedium is about 4.5 mM, about 5.5 mM, about 6.6 mM, about 7.5 mM orabout 8.5 mM. In some embodiments, the concentration of nicotinamide inthe medium is about 6.5 mM.

Additional Components

In some embodiments, the medium comprises one or more of glucose,ethanolamine, zinc sulfate, human serum, sodium selenite, ascorbic acid,and β-mercaptoethanol. In some embodiments, the medium comprises one ormore of glucose, ethanolamine, zinc sulfate, human serum, sodiumselenite, ascorbic acid, and β-mercaptoethanol in an amount additionalto any amount present in the base medium.

In various embodiments, the medium comprises glucose. In someembodiments, the media may comprise a total glucose concentration ofabout 15 mM to 40 mM. In some embodiments, the media may comprise atotal glucose concentration of about 15 mM to 35 mM, about 15 mM to 30mM, about 15 mM to 25 mM, about 15 mM to 20 mM, about 20 mM to 40 mM,about 20 mM to 35 mM, about 20 mM to 30 mM, about 20 mM to 25 mM, orabout 25 mM to 30 mM. In some embodiments, the media may comprise atotal glucose concentration of about 15 mM, about 16 mM, about 17 mM,about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM,about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM,or about 40 mM glucose. In some embodiments, the media may comprise atotal glucose concentration of about 27 mM glucose. In some embodiments,the media may comprise a total glucose concentration of about 20 mM. Inany of these embodiments, the glucose may be provided to the medium froma base media (e.g., DMEM/F12 or DMEM (high glucose)/F12) and/or may beadded to the medium in a supplement (“added glucose”). In someembodiments, the media may comprise about 2 to 40 mM of glucose providedfrom one or more commercial sources (e.g., base medium DMEM/F12 or DMEM(high glucose)/F12). In some embodiments, the medium may contain about 5to 15 mM or about 5 to 25 mM of glucose sourced from a base medium(e.g., DMEM, DMEM (high glucose), F12). In some embodiments, the mediummay comprise about 10 to 15 mM or about 10 to 25 mM of glucose sourcedfrom a base medium (e.g., DMEM/F12, DMEM (high glucose)/F12). In someembodiments, the medium may comprise 1 to 15 mM of glucose in additionto that provided in the base medium. For example, in some embodiments,an additional 1 to 15 mM or an additional 2 to 12 mM of glucose is addedto the medium. In some embodiments, an additional 1 to 15 mM, 1 to 10mM, 2 to 12 mM, 2 to 10 mM, 5 to 15 mM, or 5 to 10 mM of glucose isadded to the medium. In some embodiments, about 0.5 mM, about 1.0 mM,about 1.5 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM,about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM,about 2.9 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.1 mM,about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM,about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about 6 mM,about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 10.10 mM, about10.25 mM, about 10.5 mM, about 10.75 mM, about 11 mM, about 12 mM, about13 mM, about 14 mM, or about 15 mM of glucose is added to the medium. Insome embodiments, about 2.3 mM of glucose is added to the medium. Insome embodiments, about 4.66 mM of glucose is added to the medium. Insome embodiments, about 10.25 mM of glucose is added to the medium.

In some embodiments, ethanolamine is added to the medium at aconcentration of about 10-100 μM. In some embodiments, ethanolamine isadded to the medium at a concentration of about 50 μM. In someembodiments, zinc sulfate is added to the medium at a concentration ofabout 1.7 μM to 40 μM. In some embodiments, zinc sulfate is added to themedium at a concentration of about 20 μM to 40 μM. In some embodiments,the medium comprises about 37 μM of zinc sulfate. In some embodiments,the medium comprises about 36 μM (e.g., 36.2 μM) of zinc sulfate. Insome embodiments, the medium comprises 2% to 40% human serum. In someembodiments, the medium comprises 20-40% human serum. In someembodiments, the medium comprises 2-20% human serum. In someembodiments, the medium comprises about 15% human serum. In someembodiments, the medium comprises about 20% of human serum. In someembodiments, the medium comprises 0 μM to 50 μM β-mercaptoethanol. Insome embodiments, the medium comprises 0 μM to 7.5 μM β-mercaptoethanol.In some embodiments, the medium comprises 0 μM-5 μM β-mercaptoethanol.In some embodiments, the medium comprises 0.1 μM-5 μM β-mercaptoethanol.In some embodiments, the medium comprises about 1 μM β-mercaptoethanol.In some embodiments, the medium does not comprise β-mercaptoethanol. Insome embodiments, sodium selenite is added to the medium at aconcentration of about 1 ng/mL to 10 ng/mL. In some embodiments, themedium comprises about 5 ng/mL sodium selenite. In some embodiments,ascorbic acid is added to the medium at a concentration of about 1 to 30μg/mL. In some embodiments, the medium comprises about 15 μg/mL ofascorbic acid. In some embodiments, the medium comprises about 20 μg/mLascorbic acid.

In some embodiments, a medium described herein comprises glucose, zincsulfate, human serum, ethanolamine, and β-mercaptoethanol. In someembodiments, the medium described herein comprises (i) a totalconcentration of glucose of about 2 mM to about 40 mM; (ii) aconcentration of about 10 μM to about 100 μM of ethanolamine; (iii) aconcentration of about 1.7 μM to about 40 μM zinc sulfate; (iv) aconcentration of about 2% to 40% human serum; and/or (v) a concentrationof about 0.1 μM to 50 μM β-mercaptoethanol. In some embodiments, themedium described herein comprises glucose, zinc sulfate, human serum,ethanolamine, and β-mercaptoethanol. In some embodiments the mediumdescribed herein comprises (i) a total concentration of about 27 mM ofglucose; (ii) a concentration of about 50 μM of ethanolamine; (iii) aconcentration of about 37 μM zinc sulfate; (iv) a concentration of about15% human serum; and (v), a concentration of about 1 μMβ-mercaptoethanol.

In some embodiments, a medium described herein comprises glucose, zincsulfate, human serum, ethanolamine, sodium selenite, ascorbic acid orany combination thereof. In some embodiments, a medium described hereincomprises glucose, zinc sulfate, human serum, ethanolamine or anycombination thereof. In some embodiments, a medium described hereincomprises (i) a total concentration of about 2 mM to about 40 mM ofglucose; (ii) a concentration of about 10 μM to about 100 μM ofethanolamine; (iii) a concentration of about 1.7 μM to about 40 μM zincsulfate; and (iv) a concentration of about 2% to 40% human serum. Insome embodiments, a medium described herein comprises (i) a totalconcentration of about 20 mM of glucose; (ii) a concentration of about50 μM of ethanolamine; (iii) a concentration of about 36.2 μM or 37 μMzinc sulfate; (iv) and a concentration of about 20% human serum.

In some embodiments, a medium described herein comprises glucose, zincsulfate, human serum, ethanolamine, sodium selenite, ascorbic acid orany combination thereof. In some embodiments, a medium described hereincomprises glucose, zinc sulfate, human serum, ethanolamine or anycombination thereof. In some embodiments, a medium described hereincomprises (i) an total concentration of about 2 mM to about 40 mM ofglucose; (ii) a concentration of about 10 μM to about 100 μM ofethanolamine; (iii) a concentration of about 1.7 μM to about 40 μM zincsulfate; and (iv) a concentration of about 2% to 40% human serum. Insome embodiments, a medium described herein comprises (i) a totalconcentration of about 20 mM of glucose; (ii) a concentration of about50 μM of ethanolamine; (iii) a concentration of about 37 μM zincsulfate; (iv) and a concentration of about 15% human serum.

In some embodiments, a medium described herein comprises DMEM/F12 mediumand a supplement of glucose, zinc sulfate, human serum, ethanolamine,β-mercaptoethanol, or any combination thereof. DMEM (high glucose) Insome embodiments, the supplement provides (i) an additionalconcentration of glucose of about 2 mM to about 40 mM; (ii) anadditional concentration of about 10 μM to about 100 μM of ethanolamine;(iii) an additional concentration of about 1.7 μM to about 40 μM zincsulfate; (iv) an additional concentration of about 2% to 40% humanserum; and/or (v) an additional concentration of about 0.1 μM to 50 μMβ-mercaptoethanol. In some embodiments, the supplement provides anadditional concentration of glucose, zinc sulfate, human serum,ethanolamine, and β-mercaptoethanol. In some embodiments, the supplementprovides (i) an additional concentration of about 10.25 mM of glucose;(ii) an additional concentration of about 50 μM of ethanolamine; (iii)an additional concentration of about 37 μM zinc sulfate; (iv) anadditional concentration of about 15% human serum; and (v), anadditional concentration of about 1 μM β-mercaptoethanol.

In some embodiments, a medium described herein comprises DMEM/F12 mediumand a supplement of glucose, zinc sulfate, human serum, ethanolamine,sodium selenite, ascorbic acid or any combination thereof. DMEM (highglucose) In some embodiments, a medium described herein comprisesDMEM/F12 medium and a supplement of glucose, zinc sulfate, human serum,ethanolamine or any combination thereof. In some embodiments, thesupplement provides (i) an additional concentration of about 2 mM toabout 20 mM of glucose; (ii) an additional concentration of about 10 μMto about 100 μM of ethanolamine; (iii) an additional concentration ofabout 1.7 μM to about 40 μM zinc sulfate; and (iv) an additionalconcentration of about 2% to 40% human serum. In some embodiments, thesupplement provides (i) an additional concentration of about 4.66 mM ofglucose; (ii) an additional concentration of about 50 μM ofethanolamine; (iii) an additional concentration of about 36.2 μM or 37μM zinc sulfate; (iv) and an additional concentration of about 20% humanserum.

In some embodiments, a medium described herein comprises DMEM/F12 mediumand a supplement of glucose, zinc sulfate, human serum, ethanolamine,sodium selenite, ascorbic acid or any combination thereof. DMEM (highglucose) In some embodiments, a medium described herein comprisesDMEM/F12 medium and a supplement of glucose, zinc sulfate, human serum,ethanolamine or any combination thereof. DMEM (high glucose) In someembodiments, the supplement provides (i) an additional concentration ofabout 2 mM to about 40 mM of glucose; (ii) an additional concentrationof about 10 μM to about 100 μM of ethanolamine; (iii) an additionalconcentration of about 1.7 μM to about 40 μM zinc sulfate; and (iv) anadditional concentration of about 2% to 40% human serum. In someembodiments, the supplement provides (i) an additional concentration ofabout 2.3 mM of glucose; (ii) an additional concentration of about 50 μMof ethanolamine; (iii) an additional concentration of about 37 μM zincsulfate; (iv) and an additional concentration of about 15% human serum.

Exemplary Differentiation Medium Compositions

In some embodiments, the cells described herein are cultured in one,two, three, four, five, six, seven or eight media. In some embodiments,the cells described herein are cultured in a first medium, followed by asecond medium, a third medium, a fourth medium, a fifth medium, a sixthmedium, and a seventh medium. In some embodiments, the cells describedherein are cultured in a first medium, followed by a second medium, athird medium, a fourth medium, a fifth medium, a sixth medium, a seventhmedium and an eighth medium. In some embodiments, the cells are culturedin a medium described herein for one hour to 28 days. In someembodiments, the cells are cultured in a medium described herein for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26days, 27 days, or 28 days. In some embodiments, the cells are culturedin a medium described herein for 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18, hours, 19 hours,20 hours, 21 hours, 22 hours, or 23 hours.

In some embodiments, a method for differentiation of a population ofstem cells into a population comprising HSPCs (e.g., Stage I) utilizesthe first, second, third and fourth mediums described herein. In someembodiments, a method for differentiation of a population comprisingHSPCs into a population comprising NK cells (e.g., Stage II) utilizesthe fifth, sixth and seventh mediums described herein. In someembodiments, an alternate method for differentiation of a populationcomprising HSPCs into a population comprising NK cells (e.g., Stage II)utilizes the fifth, sixth, seventh, and eight mediums described herein.

In some embodiments, the concentrations of medium components describedherein are the total concentration of the component in the media. Insome embodiments, the concentration of the medium component describedherein is added in addition to any amount of the same component alreadypresent in the described medium. For example, a base medium containing agrowth factor may have an additional supplement of the same growthfactor added to the medium to yield a higher concentration of saidgrowth factor. Additionally, in other examples, the concentrationdescribed is the final concentration of a factor in the medium.

First Medium

In some embodiments, a population of stem cells are cultured in a firstmedium comprising any of the ROCK inhibitors described herein. In someembodiments, stem cells are cultured in the first medium comprising theROCK inhibitor thiazovivin. In some embodiments, stem cells are culturedin the ROCK inhibitor Y-27632 (TOCRIS). In some embodiments, the firstmedium comprises a concentration of about 10 μM of the ROCK inhibitor.In some embodiments, the first medium comprises StemFlex™ medium. Insome embodiments, the first medium comprises StemBrew™ Basal Media. Insome embodiments, the first medium comprises StemFlex™ Supplement. Insome embodiments, the first medium comprises StemBrew™ Supplement. Insome embodiments, the first medium comprises StemFlex™ medium and a ROCKinhibitor. In some embodiments, the first medium comprises StemBrewmedia and a ROCK inhibitor. In some embodiments, the first mediumcomprises StemFlex™ medium and thiazovivin. In some embodiments, thefirst medium comprises StemBrew media and thiazovivin. In someembodiments, the first medium comprises StemFlex™ medium and Y27632. Insome embodiments, the first medium comprises StemBrew media and Y27632.In some embodiments, the first medium comprises StemFlex™ medium and aconcentration of about 10 μM of thiazovivin. In some embodiments, thefirst medium comprises StemFlex™ medium and a concentration of about 10μm of Y27632. In some embodiments, the first medium comprises StemBrewmedium and a concentration of about 10 μM of thiazovivin. In someembodiments, the first medium comprises StemBrew medium and aconcentration of about 10 μM of Y27632.

In some embodiments, stem cells are cultured in the first medium for atime and under conditions sufficient to form stem cell aggregates. Insome embodiments, cells are cultured in the first medium for 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, or 48hours. In some embodiments, stem cells are cultured in the first mediumfor 12-48 hours. In some embodiments, stem cells are cultured in thefirst medium for 16-20 hours.

In some embodiments, the first medium comprises the composition setforth in Table 18A or Table 18B. In some embodiments, the cells arecultured in the media in Table 18A or 18B for 12-48 hours. In someembodiments, the cells are cultured in the media in Table 18A or 18B for16-20 hours.

TABLE 18A Exemplary First-Medium composition Component Working Conc.STEMFLEX ™ Basal 90% STEMFLEX ™ Supplement 1X ROCK Inhibitor 10 μM

TABLE 18B Exemplary First Medium composition Component Working Conc.StemBrew Basal Media 90% StemBrew Supplement 1X ROCK Inhibitor 10 μM

Second Medium

In some embodiments, cells are cultured in a second medium comprising abone morphogenetic protein (BMP). In some embodiments, cells arecultured in a second medium comprising a ROCK inhibitor and a BMP. Insome embodiments, cells are cultured in the second medium comprising theROCK inhibitor thiazovivin and a BMP. In some embodiments, cells arecultured in a second medium comprising the ROCK inhibitor Y27632 and aBMP. In some embodiments, cells are cultured in the second mediumcomprising a ROCK inhibitor and BMP-4. In some embodiments, cells arecultured in a second medium comprising the ROCK inhibitor Y27632 andBMP-4. In some embodiments, cells are cultured in the second mediumcomprising the ROCK inhibitor thiazovivin and BMP-4. In someembodiments, the second medium comprises a concentration of about 10 μMof the ROCK inhibitor. In some embodiments, the second medium comprisesa concentration of about 30 ng/mL of BMP-4. In some embodiments, thesecond medium comprises APEL medium. In some embodiments, the secondmedium comprises APEL medium, a ROCK inhibitor and a BMP. In someembodiments, the second medium comprises APEL medium, a ROCK inhibitorand BMP-4. In some embodiments, the second medium comprises APEL medium,Thiazovivin, and a BMP. In some embodiments, the second medium comprisesAPEL medium, Thiazovivin, and BMP-4. In some embodiments, the secondmedium comprises APEL medium, Y27632, and a BMP. In some embodiments,the second medium comprises APEL medium, Y27632, and BMP-4. In someembodiments, the second medium comprises APEL medium and a BMP. In someembodiments, the second medium comprises APEL medium and BMP-4.

In some embodiments, cells are cultured in the second medium for 1hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, or 24 hours. In some embodiments, cells are cultured in thesecond medium for up to 24 hours. In some embodiments, cells arecultured in the second medium for 6-10 hours.

In some embodiments, cells are cultured in the second medium after beingcultured in the first medium described supra.

In some embodiments, the second medium comprises the composition setforth in Table 19A or Table 19B. In some embodiments, the cells arecultured in the media in Table 19A or Table 19B for up to 24 hours. Insome embodiments, the cells are cultured in the media in Table 19A orTable 19B for 4-24 hours. In some embodiments, the cells are cultured inthe media in Table 19A or Table 19B for 6-10 hours. In some embodiments,the cells are cultured in the media in Table 19A or Table 19B afterbeing cultured in the media in Table 18A or 18B.

TABLE 19A Exemplary Second-Medium composition Component Working Conc.STEMdiff APEL 2 Medium 100% rh BMP-4 30 ng/mL ROCK Inhibitor 10 μM

TABLE 19B Exemplary Second-Medium composition Component Working Conc.STEMdiff APEL 2 Medium 100% rh BMP-4 30 ng/mL

Third Medium

In some embodiments, cells are cultured in a third medium comprisingFGF, a bone morphogenetic protein, a WNT pathway activator, andActivin-A. In some embodiments, the bone morphogenic protein in thethird medium is BMP-4. In some embodiments, the WNT pathway activator inthe third medium is CHIR-99021. In some embodiments, the FGF is bFGF orFGF-2. In some embodiments, the third medium comprises a concentrationof about 100 ng/mL of FGF. In some embodiments, the third mediumcomprises a concentration of about 30 ng/mL of BMP-4. In someembodiments, the third medium comprises a concentration of about 2.5 μMto about 3.5 μM of CHIR-99021. In some embodiments, the third mediumcomprises a concentration of about 6 μM of CHIR-99021. In someembodiments, the third medium comprises a concentration of about 7 μM ofCHIR-99021. In some embodiments, the third medium comprises aconcentration of about 5 ng/mL of Activin-A. In some embodiments, thethird medium comprises APEL medium. In some embodiments, the thirdmedium comprises APEL medium, FGF, BMP-4, CHIR-99021, and Activin A.

In some embodiments, cells are cultured in the third medium for 1-3days. In some embodiments, cells are cultured in the third medium for 1day, 2 days, or 3 days. In some embodiments, cells are cultured in thethird medium for 2 days.

In some embodiments, cells are cultured in the third medium after beingcultured in the first and second media described supra.

In some embodiments, the third medium comprises the composition setforth in Table 20A or Table 20B. In some embodiments, the cells arecultured in the third media in Table 20A or Table 20B for 1-3 Days. Insome embodiments, the cells are cultured in the third media in Table 20Aor Table 20B for about 2 days. In some embodiments, the cells arecultured in the media in Table 20A or Table 20B after being cultured inthe media in Tables 18A or 18B and 19A or 19B. In some embodiments, thecells are cultured in the third media in Table 20A after being culturedin the media in Tables 18A and 19A. In some embodiments, the cells arecultured in the third media in Table 20B after being cultured in themedia in Tables 18B and 19A. In some embodiments, the cells are culturedin the third media in Table 20A after being cultured in the media inTables 18A and 19B. In some embodiments, the cells are cultured in thethird media in Table 20B after being cultured in the media in Tables 18Band 19B.

TABLE 20A Exemplary Third-Medium composition Component Working Conc.STEMdiff APEL 2 Medium 100% rh BMP-4  30 ng/mL rh FGF2 100 ng/mLCHIR-99021 6 μM Activin-A  5 ng/mL

TABLE 20B Exemplary Third-Medium composition Component Working Conc.STEMdiff APEL 2 Medium 100% rh BMP-4  30 ng/mL rh FGF2 100 ng/mLCHIR-99021 7 μM Activin-A  5 ng/mL

Fourth Medium

In some embodiments, cells are cultured in a fourth medium comprisingFGF, VEGF, TPO, SCF, IL-3, FLT3L, a Porcn inhibitor, and anactivin/nodal inhibitor. In some embodiments, the Porcn inhibitor in thefourth medium is WNT C-59. In some embodiments, cells are cultured in afourth medium comprising FGF, VEGF, TPO, SCF, IL-3, FLT3L, and anactivin/nodal inhibitor. In some embodiments, the activin/nodalinhibitor in the fourth medium is SB-431542. In some embodiments, thefourth medium comprises a concentration of about 10-20 ng/mL of FGF. Insome embodiments, the fourth medium comprises a concentration of about20 ng/mL of FGF. In some embodiments, the fourth medium comprises aconcentration of about 20 ng/mL of VEGF. In some embodiments, the fourthmedium comprises a concentration of about 20 ng/mL of TPO. In someembodiments, the fourth medium comprises a concentration of about 100ng/mL of SCF. In some embodiments, the fourth medium comprises aconcentration of about 40 ng/mL of IL-3. In some embodiments, the fourthmedium comprises a concentration of about 20 ng/mL of FLT3L. In someembodiments, the fourth medium comprises a concentration of about 0-2 μMof WNT C-59. In some embodiments, the fourth medium comprises aconcentration of about 2 μM of WNT C-59. In some embodiments, the fourthmedium does not comprise any WNT C-59. In some embodiments, the fourthmedium comprises a concentration of about 5 μM of SB-431542. In someembodiments, the fourth medium comprises APEL medium. In someembodiments, the fourth medium comprises APEL medium, FGF, VEGF, TPO,SCF, IL-3, FLT3L, a Porcn inhibitor, and an activin/nodal inhibitor. Insome embodiments, the fourth medium comprises APEL medium, FGF, VEGF,TPO, SCF, IL-3, FLT3L, and an activin/nodal inhibitor. In someembodiments, the fourth medium comprises APEL medium, FGF, VEGF, TPO,SCF, IL-3, FLT3L, WNT C-59, and SB-431542. In some embodiments, thefourth medium comprises APEL medium, FGF, VEGF, TPO, SCF, IL-3, FLT3L,and SB-431542.

In some embodiments, cells are cultured in the fourth medium for 1-3days. In some embodiments, cells are cultured in the fourth medium for 1day, 2 days, or 3 days. In some embodiments, cells are cultured in thefourth medium for about 2 days.

In some embodiments, the fourth media comprises the composition setforth in Table 21A or 21B. In some embodiments, the cells are culturedin the fourth media in Table 21A or 21B for 1-3 days. In someembodiments, the cells are cultured in the fourth media in Table 21A or21B for about 2 days. In some embodiments, the cells are cultured in themedia in Table 21A or 21B after being cultured in the media in Tables18A/18B-20A/20B. For example, in some embodiments, the cells arecultured in the media in Table 21A after being cultured in the media inTables 18A, 19A, and 20A. In some embodiments, the cells are cultured inthe media in Table 21B after being cultured in the media in Tables 18B,19A, and 20B. For example, in some embodiments, the cells are culturedin the media in Table 21A after being cultured in the media in Tables18A, 19B, and 20A. In some embodiments, the cells are cultured in themedia in Table 21B after being cultured in the media in Tables 18B, 19B,and 20B.

TABLE 21A Exemplary Fourth-Medium composition Component Working Conc.STEMdiff APEL 2 Medium 100% rh FGF2  20 ng/mL rh VEGF165  20 ng/mL rhTPO  20 ng/mL rh SCF 100 ng/mL rh IL-3  40 ng/mL rh Flt3L  20 ng/mL WNTC-59 2 μM SB431542 5 μM

TABLE 21B Exemplary Fourth-Medium composition. Component Working Conc.STEMdiff APEL 2 Medium 100% rh FGF2  20 ng/mL rh VEGF165  20 ng/mL rhTPO  20 ng/mL rh SCF 100 ng/mL rh IL-3  40 ng/mL rh Flt3L  20 ng/mLSB431542 5 μM

Fifth Medium

In some embodiments, the cells are cultured in a fifth medium comprisingFGF, VEGF, TPO, SCF, IL-3, and FLT3L. In some embodiments, the fifthmedium comprises a concentration of about 20 ng/mL of FGF. In someembodiments, the fifth medium comprises a concentration of about 20ng/mL of VEGF. In some embodiments, the fifth medium comprises aconcentration of about 20 ng/mL of TPO. In some embodiments, the fifthmedium comprises a concentration of about 100 ng/mL of SCF. In someembodiments, the fifth medium comprises a concentration of about 40ng/mL of IL-3. In some embodiments, the fifth medium comprises aconcentration of about 10-20 ng/mL of FLT3L. In some embodiments, thefifth medium comprises a concentration of about 20 ng/mL of FLT3L Insome embodiments, the fifth medium comprises at least APEL medium. Insome embodiments, the fifth medium comprises APEL, FGF, VEGF, TPO, SCF,IL-3 and FLT3L.

In some embodiments, cells are cultured in the fifth medium for 1-3days. In some embodiments, the cells are cultured in the fifth mediumfor 1 day, 2 days, or 3 days. In some embodiments, cells are cultured inthe fifth medium for about 2 days.

In some embodiments, the fifth medium comprises the composition setforth in Table 22. In some embodiments, the cells are cultured in thefifth medium in Table 22 for 2 days. In some embodiments, the cells arecultured in the fifth media in Table 22 for about 2 days. In someembodiments, the cells are cultured in the media in Table 22 after beingcultured in the media in Tables 18A, 19A, 20A, and 21A. In someembodiments, the cells are cultured in the media in Table 22 after beingcultured in the media in Tables 18B, 19A, 20B, and 21B. In someembodiments, the cells are cultured in the media in Table 22 after beingcultured in the media in Tables 18A, 19B, 20A, and 21A. In someembodiments, the cells are cultured in the media in Table 22 after beingcultured in the media in Tables 18B, 19B, 20B, and 21B.

TABLE 22 Exemplary Fifth-Medium composition Component Working Conc.STEMdiff APEL 2 Medium 100% rh FGF2  20 ng/mL rh VEGF165  20 ng/mL rhTPO  20 ng/mL rh SCF 100 ng/mL rh IL-3  40 ng/mL rh Flt3L  20 ng/mL

Sixth Medium

In some embodiments, cells are cultured in a sixth medium comprisingIL-3, IL-7, FLT3L, IL-15, and SCF. In some embodiments, the sixth mediumcomprises a concentration of about 5 ng/mL of IL-3. In some embodiments,the sixth medium comprises a concentration of about 20 ng/mL of IL-7. Insome embodiments, the sixth medium comprises a concentration of about10-20 ng/mL of FLT3L. In some embodiments, the sixth medium comprises aconcentration of about 15 ng/mL of FLT3L. In some embodiments, the sixthmedium comprises a concentration of about 10-20 ng/mL of IL-15. In someembodiments, the sixth medium comprises a concentration of about 15ng/mL of IL-15. In some embodiments, the sixth medium comprises aconcentration of about 20 ng/mL of SCF. In some embodiments, the sixthmedium comprises a concentration of about 20 ng/mL of FLT3L.

In some embodiments, the sixth medium comprises human serum, zincsulfate, ethanolamine, β-mercaptoethanol, glucose, and glutamax (e.g.,glutamine substitute) in addition to any amounts present in the basemedium. In some embodiments, the sixth medium comprises about 15% humanserum. In some embodiments, the sixth medium comprises a concentrationof about 37 μM of zinc sulfate. In some embodiments, the sixth mediumcomprises a concentration of about 50 μM of ethanolamine. In someembodiments, the sixth medium comprises a concentration of about 1 μM ofβ-mercaptoethanol. In some embodiments, the sixth medium comprises atotal concentration of about 27 mM of glucose. This concentration isinclusive of glucose sourced from other components in the medium (e.g.,DMEM, DMEM (high glucose), and/or F-12 supplement) as well as additionalglucose added to the medium (“added glucose”). In some cases, the sixthmedium comprises about 1 to 15 mM of “added glucose.” In some cases, thesixth medium comprises about 10.25 mM of “added glucose.” In someembodiments, the sixth medium comprises a concentration of 1× glutamax.

In some embodiments, the sixth medium comprises human serum, zincsulfate, ethanolamine, glucose, and glutamax in addition to any amountspresent in the base medium. In some embodiments, the sixth mediumcomprises about 20% human serum. In some embodiments, the sixth mediumcomprises a concentration of about 36.2 μM of zinc sulfate. In someembodiments, the sixth medium comprises a concentration of about 50 μMof ethanolamine. In some embodiments, the sixth medium comprises a totalconcentration of about 20 mM of glucose. This concentration is inclusiveof glucose sourced from other components in the medium (e.g., DMEM, DMEM(high glucose), and/or F-12 supplement) as well as additional glucoseadded to the medium (“added glucose”). In some cases, the sixth mediumcomprises about 1 to 5 mM of “added glucose.” In some cases, the sixthmedium comprises about 4.66 mM of “added glucose.” In some embodiments,the sixth medium comprises a concentration of 1× glutamax.

In some embodiments, the sixth medium comprises DMEM/F12 medium. In someembodiments, DMEM/F12 medium is the base medium. In some embodiments,the sixth medium comprises DMEM/F12 medium, IL-3, IL-7, FLT3L, IL-15,and SCF. In some embodiments, the sixth medium comprises DMEM/F12medium, IL-3, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate,ethanolamine, β-mercaptoethanol, glucose, and glutamax. In someembodiments, the sixth medium comprises DMEM/F12 medium, IL-3, IL-7,FLT3L, IL-15, SCF, human serum, zinc sulfate, ethanolamine, glucose, andglutamax.

In some embodiments, the sixth medium comprises DMEM (high glucose)/F12medium. In some embodiments, DMEM (high glucose)/F12 medium is the basemedium. In some embodiments, the sixth medium comprises DMEM (highglucose)/F12 medium, IL-3, IL-7, FLT3L, IL-15, and SCF. In someembodiments, the sixth medium comprises DMEM (high glucose)/F12 medium,IL-3, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate, ethanolamine,β-mercaptoethanol, glucose, and glutamax. In some embodiments, the sixthmedium comprises DMEM (high glucose)/F12 medium, IL-3, IL-7, FLT3L,IL-15, SCF, human serum, zinc sulfate, ethanolamine, glucose, andglutamax.

In some embodiments, cells are cultured in the sixth medium for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days or 8 days. In someembodiments, the cells are cultured in the sixth medium for up to 8days. In some embodiments, the cells are cultured in the sixth mediumfor 6-8 days. In some embodiments, the cells are cultured in the sixthmedium for 8 days.

In some embodiments, the sixth medium comprises the composition setforth in Table 23A or 23B. In some embodiments, the cells are culturedin the sixth media in Table 23A or 23B for up to 8 days. In someembodiments, the cells are cultured in the sixth media in Table 23A or23B for 6-8 days. In some embodiments, the cells are cultured in themedia in Table 23A or 23B after being cultured in the media in Tables18A/18B-22. For example, in some embodiments, the cells are cultured inthe media in Table 23A after being cultured in the media in Tables 18A,19A, 20A, 21A, and 22. In additional embodiments, the cells are culturedin the media in Table 23B after being cultured in the media in Tables18B, 19A, 20B, 21B and 22. In some embodiments, the cells are culturedin the media in Table 23A after being cultured in the media in Tables18A, 19B, 20A, 21A, and 22. In additional embodiments, the cells arecultured in the media in Table 23B after being cultured in the media inTables 18B, 19B, 20B, 21B and 22.

TABLE 23A Exemplary Sixth-Medium composition Component Working Conc.DMEM (high glucose, GlutaMAX) 55.47% F-12 with GlutaMAX 27.74% GlutaMAX1X Glucose* 10.25 mM Human AB serum   15% Zinc sulfate 37 μMEthanolamine 50 μM Ascorbic acid 20 μg/mL Sodium selenite  5 ng/mLβ-mercaptoethanol  1 μM rh IL-3  5 ng/mL rh IL-7 20 ng/mL rh Flt3L 15ng/mL rh IL-15 15 ng/mL rh SCF 20 ng/mL *Total glucose concentration inmedium is 27 mM (accounting for glucose in DMEM (high glucose) medium,F12 supplement and any added glucose).

TABLE 23B Exemplary Sixth Medium. Component Working Conc. DMEM (highglucose, GlutaMAX) 50.3% F-12 with GlutaMAX   28% GlutaMAX 1X Glucose*4.66 mM Human AB serum   20% Zinc sulfate 36.2 μM Ethanolamine   50 μMAscorbic acid 15 μg/mL Sodium selenite  5 ng/mL rh IL-3  5 ng/mL rh IL-720 ng/mL rh Flt3L 15 ng/mL rh IL-15 15 ng/mL rh SCF 20 ng/mL *Totalglucose concentration in medium is 20 mM (accounting for glucose in DMEM(high glucose) medium, F12 supplement and any added glucose).

Seventh Medium

In some embodiments, cells are cultured in a seventh medium comprisingIL-7, FLT3L, IL-15, and SCF. In some embodiments, the seventh mediumcomprises a concentration of about 20 ng/mL of IL-7. In someembodiments, the seventh medium comprises a concentration of about 10-20ng/mL of FLT3L. In some embodiments, the seventh medium comprises aconcentration of about 15 ng/mL of FLT3L. In some embodiments, theseventh medium comprises a concentration of about 10-20 ng/mL of IL-15.In some embodiments, the seventh medium comprises a concentration ofabout 15 ng/mL of IL-15. In some embodiments, the seventh mediumcomprises a concentration of about 20 ng/mL of SCF.

In some embodiments, the seventh medium comprises human serum, zincsulfate, ethanolamine, β-mercaptoethanol, glucose, and glutamax inaddition to any amounts present in the base medium. In some embodiments,the seventh medium comprises about 15% human serum. In some embodiments,the seventh medium comprises a concentration of about 37 μM of zincsulfate. In some embodiments, the seventh medium comprises aconcentration of about 50 μM of ethanolamine. In some embodiments, theseventh medium comprises a concentration of about 1 μM ofβ-mercaptoethanol. In some embodiments, the seventh medium comprises atotal concentration of about 27 mM of glucose. This concentration isinclusive of glucose sourced from other components in the medium (e.g.,DMEM, DMEM (high glucose), and/or F-12 supplement) as well as additionalglucose added to the medium (“added glucose”). In some cases, theseventh medium comprises about 1 to 15 mM of “added glucose.” In somecases, the seventh medium comprises about 10.25 mM of “added glucose.”In some embodiments, the seventh medium comprises a concentration of 1×glutamax.

In some embodiments, the seventh medium comprises human serum, zincsulfate, ethanolamine, glucose, and glutamax in addition to any amountspresent in the base medium. In some embodiments, the seventh mediumcomprises about 20% human serum. In some embodiments, the seventh mediumcomprises a concentration of about 37 μM of zinc sulfate. In someembodiments, the seventh medium comprises a concentration of about 50 μMof ethanolamine. In some embodiments, the seventh medium comprises atotal concentration of about 20 mM of glucose. This concentration isinclusive of glucose sourced from other components in the medium (e.g.,DMEM, DMEM (high glucose), and/or F-12 supplement) as well as additionalglucose added to the medium (“added glucose”). In some cases, theseventh medium comprises about 1 to 5 mM of “added glucose.” In somecases, the seventh medium comprises about 4.66 mM of “added glucose.” Insome embodiments, the seventh medium comprises a concentration of 1×glutamax.

In some embodiments, the seventh medium comprises DMEM/F12 medium. Insome embodiments, DMEM/F12 medium is the base medium. In someembodiments, the seventh medium comprises DMEM/F12 medium, IL-7, FLT3L,IL-15, and SCF. In some embodiments, the seventh medium comprisesDMEM/F12 medium, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate,ethanolamine, β-mercaptoethanol, glucose, and glutamax. In someembodiments, the seventh medium comprises DMEM/F12 medium, IL-7, FLT3L,IL-15, SCF, human serum, zinc sulfate, ethanolamine, glucose, andglutamax.

In some embodiments, the seventh medium comprises DMEM (highglucose)/F12 medium. In some embodiments, DMEM (high glucose)/F12 mediumis the base medium. In some embodiments, the seventh medium comprisesDMEM (high glucose)/F12 medium, IL-7, FLT3L, IL-15, and SCF. In someembodiments, the seventh medium comprises DMEM (high glucose)/F12medium, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate,ethanolamine, β-mercaptoethanol, glucose, and glutamax. In someembodiments, the seventh medium comprises DMEM (high glucose)/F12medium, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate,ethanolamine, glucose, and glutamax.

In some embodiments, the cells are cultured in the seventh medium for upto 6 days. In some embodiments, the cells are cultured in the seventhmedium for 6 days. In some embodiments, the cells are cultured in theseventh medium for 14 days. In some embodiments, the cells are culturedin the seventh medium for at least 6 days and up to 21 to 28 days. Insome embodiments, the cells are cultured in the seventh medium for 6-28days.

In some embodiments, the seventh medium comprises the composition setforth in Table 24A or 24B. In some embodiments, the cells are culturedin the seventh media in Table 24A or 24B for at least 6 days. In someembodiments, the cells are cultured in the seventh media in Table 24Afor 6-28 days. In some embodiments, the cells are cultured in theseventh media in Table 24A for 14-28 days. In some embodiments, thecells are cultured in the seventh media in Table 24A for up to 14 days,up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25days, up to 26 days, up to 27 days, or up to 28 days. In someembodiments, the cells are cultured in the seventh media in Table 24Bfor up to 6 days. In some embodiments, the cells are cultured in theseventh media in Table 24B for 6 days. In some embodiments, the cellsare cultured in the media in Table 24A or 24B after being cultured inthe media in Tables 18A-23B. For example, the cells can be cultured inthe media in Table 24A after being cultured in the media in Tables 18A,19A, 20A, 21A, 22 and 23A. As another example, the cells can be culturedin the media in Table 24B after being cultured in the media in Tables18B, 19A, 20B, 21B, 22 and 23B. For example, the cells can be culturedin the media in Table 24A after being cultured in the media in Tables18A, 19B, 20A, 21A, 22 and 23A. As another example, the cells can becultured in the media in Table 24B after being cultured in the media inTables 18B, 19B, 20B, 21B, 22 and 23B.

TABLE 24A Exemplary Seventh-Medium composition. Component Working Conc.DMEM (high glucose, GlutaMAX) 55.47% F-12 with GlutaMAX 27.74% GlutaMAX1X Glucose* 10.25 mM Human AB serum   15% Zinc sulfate 37 μMEthanolamine 50 μM Ascorbic acid 20 μg/mL Sodium selenite  5 ng/mLβ-mercaptoethanol  1 μM rh IL-7 20 ng/mL rh Flt3L 15 ng/mL rh IL-15 15ng/mL rh SCF 20 ng/mL *Total glucose concentration in medium is 27 mM(accounting for glucose in DMEM (high glucose) medium, F12 supplementand any added glucose).

TABLE 24B Exemplary Seventh Medium composition. Component Working Conc.DMEM (high glucose, GlutaMAX) 50.3% F-12 with GlutaMAX   28% GlutaMAX 1XGlucose 4.66 mM Human AB serum   20% Zinc sulfate 37 μM Ethanolamine 50μM Ascorbic acid 15 μg/mL Sodium selenite  5 ng/mL rh IL-7 20 ng/mL rhFlt3L 15 ng/mL rh IL-15 15 ng/mL rh SCF 20 ng/mL *Total glucoseconcentration in medium is 20 mM (accounting for glucose in DMEM (highglucose) medium, F12 supplement and any added glucose).

Eighth Medium

In some embodiments, cells are cultured in an eighth medium comprisingIL-7, FLT3L, IL-15, SCF and nicotinamide. In some embodiments, cells arecultured in an eighth medium comprising IL-7, FLT3L, IL-15, and SCF. Insome embodiments, the eighth medium comprises a concentration of about10 ng/mL of IL-7. In some embodiments, the eighth medium comprises aconcentration of about 5-20 ng/mL of FLT3L. In some embodiments, theeighth medium comprises a concentration of about 7.5 ng/mL of FLT3L. Insome embodiments, the eighth medium comprises a concentration of about10-40 ng/mL of IL-15. In some embodiments, the eighth medium comprises aconcentration of about 15 ng/mL of IL-15. In some embodiments, theeighth medium comprises a concentration of about 20 ng/mL of SCF. Insome embodiments, the eighth medium comprises about 5 to 10 mM ofnicotinamide. In some embodiments, the eight medium comprises about 6.5mM of nicotinamide. In some embodiments, the eighth medium does notcomprise any nicotinamide.

In some embodiments, the eighth medium comprises human serum, zincsulfate, ethanolamine, glucose, and glutamax in addition to any amountspresent in the base medium. In some embodiments, the eighth mediumcomprises about 10% human serum. In some embodiments, the eighth mediumcomprises a concentration of about 37 μM of zinc sulfate. In someembodiments, the eighth medium comprises a concentration of about 50 μMof ethanolamine. In some embodiments, the eighth medium comprises atotal concentration of about 20 mM of glucose. This concentration isinclusive of glucose sourced from other components in the medium (e.g.,DMEM, DMEM (high glucose), and/or F-12 supplement) as well as additionalglucose added to the medium (“added glucose”). In some cases, the eighthmedium comprises about 1 to 5 mM of “added glucose.” In some cases, theeighth medium comprises about 2.3 mM of “added glucose.” In someembodiments, the eighth medium comprises a concentration of 1× glutamax.

In some embodiments, the eighth medium comprises DMEM/F12 medium. Insome embodiments, DMEM/F12 medium is the base medium. In someembodiments, the eighth medium comprises DMEM/F12 medium, IL-7, FLT3L,IL-15, SCF and nicotinamide. In some embodiments, the eighth mediumcomprises DMEM/F12 medium, IL-7, FLT3L, IL-15, and SCF. In someembodiments, the eighth medium comprises DMEM/F12 medium, IL-7, FLT3L,IL-15, SCF, nicotinamide, human serum, zinc sulfate, ethanolamine,glucose, and glutamax. In some embodiments, the eighth medium comprisesDMEM/F12 medium, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate,ethanolamine, glucose, and glutamax. In some embodiments, the eighthmedium does not comprise nicotinamide.

In some embodiments, the eighth medium comprises DMEM (high glucose)/F12medium. In some embodiments, DMEM (high glucose)/F12 medium is the basemedium. In some embodiments, the eighth medium comprises DMEM (highglucose)/F12 medium, IL-7, FLT3L, IL-15, SCF and nicotinamide. In someembodiments, the eighth medium comprises DMEM (high glucose)/F12 medium,IL-7, FLT3L, IL-15, and SCF. In some embodiments, the eighth mediumcomprises DMEM (high glucose)/F12 medium, IL-7, FLT3L, IL-15, SCF,nicotinamide, human serum, zinc sulfate, ethanolamine, glucose, andglutamax. In some embodiments, the eighth medium comprises DMEM (highglucose)/F12 medium, IL-7, FLT3L, IL-15, SCF, human serum, zinc sulfate,ethanolamine, glucose, and glutamax. In some embodiments, the eighthmedium does not comprise nicotinamide.

In some embodiments, the cells are cultured in the eighth medium for atleast 6 days and up to 10 to 16 days total. In some embodiments, thecells are cultured in the eighth medium for 8 days. In some embodiments,the cells are cultured in the eighth medium for 8 to 16 days.

In some embodiments, the eighth medium comprises the composition setforth in Table 25A or Table 25B. In some embodiments, the cells arecultured in the eighth media in Table 25A or Table 25B for at least 6days. In some embodiments, the cells are cultured in the eighth media inTable 25A or Table 25B for 8 days. In some embodiments, the cells arecultured in the eighth media in Table 25A or Table 25B for 8-28 days. Insome embodiments, the cells are cultured in the media in Table 25A orTable 25B after being cultured in the media in Tables 18B, 19A, 20B,21B, 22, 23B, and 24B. In some embodiments, the cells are cultured inthe media in Table 25A or Table 25B after being cultured in the media inTables 18B, 19B, 20B, 21B, 22, 23B, and 24B.

TABLE 25A Exemplary Eighth-Medium composition Component Working Conc.DMEM (high glucose, GlutaMAX) 60.5% F-12 with GlutaMAX   28% GlutaMAX 1XGlucose* 2.3 mM Human AB serum   10% Zinc sulfate 37 μM Ethanolamine 50μM Ascorbic acid  15 μg//mL Sodium selenite   5 ng/mL Nicotinamide 6.5mM rh IL-7  10 ng/mL rh Flt3L 7.5 ng/mL rh IL-15  15 ng/mL rh SCF  20ng/mL *Total glucose concentration in medium is 20 mM (accounting forglucose in DMEM (high glucose) medium, F12 supplement and any addedglucose).

TABLE 25B Exemplary Eighth-Medium composition Component Working Conc.DMEM (high glucose, GlutaMAX) 60.5% F-12 with GlutaMAX   28% GlutaMAX 1XGlucose* 2.3 mM Human AB serum   10% Zinc sulfate 37 μM Ethanolamine 50μM Ascorbic acid  15 μg//mL Sodium selenite   5 ng/mL rh IL-7  10 ng/mLrh Flt3L 7.5 ng/mL rh IL-15  15 ng/mL rh SCF  20 ng/mL *Total glucoseconcentration in medium is 20 mM (accounting for glucose in DMEM (highglucose) medium, F12 supplement and any added glucose).

Exemplary Differentiation Methods

Provided herein, in some embodiments, are methods for generating HSPCsfrom stem cells (e.g., iPSCs). In some embodiments, the method includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to form apopulation comprising cell aggregates;

(b) culturing the population comprising aggregates in a second mediumcomprising an amount of BMP-4, and optionally an amount of a ROCKinhibitor;

(c) culturing the population comprising aggregates in a third mediumcomprising an amount of BMP-4, FGF2, a WNT pathway activator, andActivin A;

(d) culturing the population comprising aggregates in a fourth mediumcomprising an amount of FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 andan activin/nodal inhibitor to form a cell population comprising HSPCs.

In some embodiments, steps (a)-(d) occurs between 4-9 days. In someembodiments, the cell population is cultured in step (a) for 12-48hours. In some embodiments, the population comprising aggregates iscultured in step (b) for up to 24 hours. In some embodiments, thepopulation comprising aggregates is cultured in step (c) for 1-3 days.In some embodiments, the population comprising aggregates is cultured instep (d) for 1-3 days. In some embodiments, the cell population iscultured in step (a) for 16-20 hours; the population comprisingaggregates is cultured in step (b) for 6-10 hours; the populationcomprising aggregates is cultured in step (c) for 2 days; and thepopulation comprising aggregates is cultured in step (d) for 2 days.

Provided herein, in some embodiments, are alternative methods forgenerating HSPCs from stem cells (e.g., iPSCs). In some embodiments, themethod includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to form apopulation comprising cell aggregates;

(b) culturing the population comprising aggregates in a second mediumcomprising an amount of BMP-4, and optionally an amount of a ROCKinhibitor;

(c) culturing the population comprising aggregates in a third mediumcomprising an amount of BMP-4, FGF2, a WNT pathway activator, andActivin A;

(d) culturing the population comprising aggregates in a fourth mediumcomprising an amount of FGF2, VEGF, TPO, SCF, IL-3, FLT3L and anactivin/nodal inhibitor to form a cell population comprising HSPCs.

In some embodiments, steps (a)-(d) occurs between 4-9 days. In someembodiments, the cell population is cultured in step (a) for 12-48hours. In some embodiments, the population comprising aggregates iscultured in step (b) for up to 24 hours. In some embodiments, thepopulation comprising aggregates is cultured in step (c) for 1-3 days.In some embodiments, the population comprising aggregates is cultured instep (d) for 1-3 days. In some embodiments, the cell population iscultured in step (a) for 16-20 hours; the population comprisingaggregates is cultured in step (b) for 6-10 hours; the populationcomprising aggregates is cultured in step (c) for 2 days; and thepopulation comprising aggregates is cultured in step (d) for 2 days.

Provided herein, in some embodiments, are methods for generating NaturalKiller (NK) cells from stem cells. In some embodiments, the methodincludes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to form apopulation comprising cell aggregates;

(b) culturing the population comprising aggregates in a second mediumcomprising an amount of BMP-4, and optionally an amount of a ROCKinhibitor;

(c) culturing the population comprising aggregates in a third mediumcomprising an amount of BMP-4, FGF2, a WNT pathway activator, andActivin A;

(d) culturing the population comprising aggregates in a fourth mediumcomprising an amount of FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 andan activin/nodal inhibitor to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for a time sufficient to generate NKcells. In some embodiments, the second medium further includes a ROCKinhibitor. In some embodiments, the ROCK inhibitor is thiazovivin. Insome embodiments, the ROCK inhibitor is Y27632. In some embodiments, theWNT pathway activator is CHIR-99021. In some embodiments, theactivin/nodal inhibitor is SB-431542.

In some embodiments, steps (a)-(g) occurs between 20-35 days. In someembodiments steps (a)-(g) occur in less than 20 days. In someembodiments, the cell population is cultured in step (a) for 12-48hours. In some embodiments, the population comprising aggregates iscultured in step (b) for up to 24 hours. In some embodiments, thepopulation comprising aggregates is cultured in step (c) for 1-3 days.In some embodiments, the population comprising aggregates is cultured instep (d) for 1-3 days. In some embodiments, the cell population iscultured in step (e) for 1-3 days. In some embodiments, the cellpopulation is cultured in step (f) for up to 7 days. In someembodiments, the cell population is cultured in step (g) for at least 6days and up to 21-28 days total. In some embodiments, the cellpopulation is cultured in step (a) for 16-20 hours; the populationcomprising aggregates is cultured in step (b) for 6-10 hours; thepopulation comprising aggregates is cultured in step (c) for 2 days; thepopulation comprising aggregates is cultured in step (d) for 2 days; thecell population is cultured in step (e) for 2 days; the cell populationis cultured in step (f) for 4 days; and/or the cell population iscultured in step (g) for 14-28 days.

In some embodiments, the method is carried out under suspensionagitation. In some embodiments, the suspension agitation includesrotation. In some embodiments, the first and second media includeStemFlex medium. In some embodiments, the third, fourth and fifth mediainclude APEL medium. In some embodiments, the sixth and seventh mediacomprise DMEM/F12 medium. In some embodiments, the sixth and seventhmedia comprise DMEM with high glucose and GlutaMAX (Thermo Fisher,10566016). In some embodiments, the sixth and seventh media compriseF-12 with GlutaMAX (Thermo Fisher, 31765035). In some embodiments, thesixth and seventh media include human serum, zinc sulfate, ethanolamine,β-mercaptoethanol, glucose, or any combination thereof. In someembodiments, the sixth medium comprises about 15% of human AB serum. Insome embodiments, the sixth medium comprises about 37 μM of zincsulfate. In some embodiments, the sixth medium comprises a concentrationof about 50 μM of ethanolamine. In some embodiments, the sixth mediumcomprises about 20 μg/mL of ascorbic acid. In some embodiments, thesixth medium comprises about 5 ng/mL of sodium selenite. In someembodiments, the sixth medium comprises a concentration of about 1 μM ofβ-mercaptoethanol. In some embodiments, the sixth medium comprises aconcentration of about 27 mM of glucose. In some embodiments, the sixthmedium comprises a concentration of about 27 mM of glucose, includingabout 10.25 mM of added glucose (above glucose in DMEM, DMEM (highglucose), or F12 media). In some embodiments, the seventh mediumcomprises about 15% human serum. In some embodiments, the seventh mediumcomprises a concentration of about 37 μM of zinc sulfate. In someembodiments, the seventh medium comprises a concentration of about 50 μMof ethanolamine. In some embodiments, the seventh medium comprises aconcentration of about 1 μM of β-mercaptoethanol. In some embodiments,the seventh medium comprises a concentration of about 27 mM of glucose.In some embodiments, the seventh medium comprises a concentration ofabout 27 mM of glucose, including about 10.25 mM of added glucose (aboveglucose in DMEM, DMEM (high glucose), or F12 media). In someembodiments, the seventh medium comprises a concentration of 1×glutamax.

In some embodiments, the first medium includes 10 μM of the ROCKinhibitor. In some embodiments, the second medium includes 30 ng/mLBMP-4 and 10 μM of a ROCK inhibitor. In some embodiments, the thirdmedium includes 30 ng/mL BMP-4, 100 ng/mL FGF2, 6 μM CHIR-99021, and2.5-5 ng/mL Activin A.

In some embodiments, half of the third medium is added to the stem cellaggregates. In some embodiments, the fourth and fifth media include 20ng/mL FGF, 20 ng/mL VEGF, 20 ng/mL TPO, 100 ng/mL SCF, 40 ng/mL IL-3,and 10-20 ng/mL FLT3L. In some embodiments, the fourth medium furtherincludes 2 μM WNT C-59 and 5 μM SB-431542. In some embodiments, thesixth and seventh media includes 20 ng/mL IL-7, 10-20 ng/mL FLT3L, 10-20ng/mL IL-15, and 20 ng/mL SCF. In some embodiments, the sixth mediumincludes 5 ng/mL IL-3.

Provided herein, in some embodiments, are alternative methods forgenerating NK cells from stem cells. In some embodiments, the methodincludes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to form apopulation comprising cell aggregates;

(b) culturing the population comprising aggregates in a second mediumcomprising an amount of BMP-4, and optionally an amount of a ROCKinhibitor;

(c) culturing the population comprising aggregates in a third mediumcomprising an amount of BMP-4, FGF2, a WNT pathway activator, andActivin A;

(d) culturing the population comprising aggregates in a fourth mediumcomprising an amount of FGF2, VEGF, TPO, SCF, IL-3, FLT3L, and anactivin/nodal inhibitor to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF;

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF; and;

(h) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, SCF and, optionally, nicotinamide for atime sufficient to generate NK cells.

In some embodiments, the second medium further includes a ROCKinhibitor. In some embodiments, the ROCK inhibitor is thiazovivin. Insome embodiments, the ROCK inhibitor is Y27632. In some embodiments, theWNT pathway activator is CHIR-99021. In some embodiments, theactivin/nodal inhibitor is SB-431542.

In some embodiments, steps (a)-(g) occurs between 20-35 days. In someembodiments, steps (a)-(h) occurs between 20-35 days. In someembodiments, steps (a)-(h) occurs between 23-40 days. In someembodiments, steps (a)-(h) occurs between 23-30 days. In someembodiments, the cell population is cultured in step (a) for about 12-48hours. In some embodiments, the population comprising aggregates iscultured in step (b) for up to about 24 hours. In some embodiments, thepopulation comprising aggregates is cultured in step (c) for about 1-3days. In some embodiments, the population comprising aggregates iscultured in step (d) for about 1-3 days. In some embodiments, the cellpopulation is cultured in step (e) for about 1-3 days. In someembodiments, the cell population is cultured in step (f) for up to about8 days. In some embodiments, the cell population is cultured in step (g)for up to about 6 days. In some embodiments, the cell population iscultured in step (h) for at least about 8 days and up to 10-16 daystotal. In some embodiments, the cell population is cultured in step (a)for about 16-20 hours; the population comprising aggregates is culturedin step (b) for about 6-10 hours; the population comprising aggregatesis cultured in step (c) for about 2 days; the population comprisingaggregates is cultured in step (d) for about 2 days; the cell populationis cultured in step (e) for about 2 days; the cell population iscultured in step (f) for about 8 days; the cell population is culturedin step (g) for about 6 days; and/or the cell population is cultured instep (h) for about 8 days.

In some embodiments, the method is carried out under suspensionagitation. In some embodiments, the suspension agitation includesrotation. In some embodiments, the first and second media includeStemBrew medium. In some embodiments, the first media includes StemBrewmedium. In some embodiments, the third, fourth and fifth media includeAPEL medium. In some embodiments, the second, third, fourth and fifthmedia include APEL medium. In some embodiments, the sixth, seventh andeighth media comprise DMEM/F12 medium. In some embodiments, the sixth,seventh and eighth media comprise DMEM with high glucose and GlutaMAX(Thermo Fisher, 10566016). In some embodiments, the sixth, seventh andeighth media comprise F-12 with GlutaMAX (Thermo Fisher, 31765035).

In some embodiments, the sixth, seventh and eighth media include humanserum, zinc sulfate, ethanolamine, glucose, or any combination thereof.In some embodiments, the sixth medium comprises about 20% of human ABserum. In some embodiments, the sixth medium comprises about 36.2 μM ofzinc sulfate. In some embodiments, the sixth medium comprises aconcentration of about 50 μM of ethanolamine. In some embodiments, thesixth medium comprises about 15 μg/mL of ascorbic acid. In someembodiments, the sixth medium comprises about 5 ng/mL of sodiumselenite. In some embodiments, the seventh medium comprises a totalconcentration of about 20 mM of glucose. In some embodiments, theseventh medium comprises a concentration of about 20 mM of glucose,including about 4.66 mM of added glucose (above glucose in DMEM, DMEM(high glucose), or F12 media). In some embodiments, the seventh mediumcomprises about 20% human serum. In some embodiments, the seventh mediumcomprises a concentration of about 37 μM of zinc sulfate. In someembodiments, the seventh medium comprises a concentration of about 50 μMof ethanolamine. In some embodiments, the seventh medium comprises atotal concentration of about 20 mM of glucose. In some embodiments, theseventh medium comprises a concentration of about 20 mM of glucose,including about 4.66 mM of added glucose (above glucose in DMEM, DMEM(high glucose), or F12 media). In some embodiments, the seventh mediumcomprises a concentration of 1× glutamax. In some embodiments, theeighth medium comprises about 10% human serum. In some embodiments, theeighth medium comprises a concentration of about 37 μM of zinc sulfate.In some embodiments, the eighth medium comprises a concentration ofabout 50 μM of ethanolamine. In some embodiments, the eighth mediumcomprises a total concentration of about 20 mM of glucose. In someembodiments, the eighth medium comprises a concentration of about 20 mMof glucose, including about 2.3 mM of added glucose (above glucose inDMEM, DMEM (high glucose), or F12 media). In some embodiments, theeighth medium comprises a concentration of 1× glutamax. In someembodiments, the eighth medium comprises nicotinamide. In someembodiments, the eighth medium comprises a concentration of about 1-10mM of nicotinamide. In some embodiments, the eighth medium comprises aconcentration of about 6.5 mM nicotinamide. In some embodiments, theeighth medium does not comprise nicotinamide.

In some embodiments, the first medium includes 10 μM of the ROCKinhibitor. In some embodiments, the second medium includes 30 ng/mLBMP-4 and 10 μM of a ROCK inhibitor. In some embodiments, the secondmedium includes 30 ng/mL BMP-4. In some embodiments, the third mediumincludes 30 ng/mL BMP-4, 100 ng/mL FGF2, 7 μM CHIR-99021, and 2.5-5ng/mL Activin A.

In some embodiments, half of the third medium is added to the stem cellaggregates. In some embodiments, the fourth and fifth media include 20ng/mL FGF, 20 ng/mL VEGF, 20 ng/mL TPO, 100 ng/mL SCF, 40 ng/mL IL-3,and 10-20 ng/mL FLT3L. In some embodiments, the fourth medium furtherincludes 5 μM SB-431542. In some embodiments, the sixth and seventhmedia includes 20 ng/mL IL-7, 10-20 ng/mL FLT3L, 10-20 ng/mL IL-15, and20 ng/mL SCF. In some embodiments, the sixth medium includes 5 ng/mLIL-3.

In some embodiments, the eighth medium comprises 10-20 ng/mL IL-7, 5-20ng/mL FLT3L, 10-40 ng/mL IL-15, and 20-40 ng/mL of SCF. In someembodiments, the eighth medium comprises 10 ng/mL IL-7, 7.5 ng/mL FLT3L,15 ng/mL IL-15, and 20 ng/mL of SCF. In some embodiments, the eighthmedium comprises 20 ng/mL IL-7, 15 ng/mL FLT3L, 30 ng/mL IL-15 and 40ng/mL of SCF. In some embodiments, about 50 mL of the eighth mediumcomprising high amounts of IL-7, FLT3L, IL-15 and SCF (e.g., 20 ng/mLIL-7, 15 ng/mL FLT3L, 30 ng/mL IL-15 and 40 ng/mL of SCF) replaces theeighth medium comprising low amounts of IL-7, FLT3L, IL-15, and SCF(e.g., 10 ng/mL IL-7, 7.5 ng/mL FLT3L, 15 ng/mL IL-15, and 20 ng/mL ofSCF). In some embodiments, the eighth medium includes 1-10 mM ofnicotinamide. In some embodiments, the eighth medium comprises 6.5 mM ofnicotinamide. In some embodiments, the eighth medium does not comprisenicotinamide.

In some embodiments, a method for differentiating stem cells into HSPCsincludes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days; and

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and an activin/nodalinhibitor for 1-3 days to form a cell population comprising HSPCs.

In some embodiments, an alternative method for differentiating stemcells into HSPCs includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days; and

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L and an activin/nodal inhibitor for 1-3days to form a cell population comprising HSPCs.

In some embodiments, a method for differentiating stem cells into HSPCsincludes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;and

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and an activin/nodalinhibitor for about 2 days to form a cell population comprising HSPCs.

In some embodiments, an alternative method for differentiating stemcells into HSPCs includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;and

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L and an activin/nodal inhibitor forabout 2 days to form a cell population comprising HSPCs.

In some embodiments, a method for differentiating stem cells into HSPCsincludes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL; and

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, WNT C-59 at a concentration ofabout 2 μM and SB-431542 at a concentration of about 5 μM to form a cellpopulation comprising HSPCs.

In some embodiments, an alternative method for differentiating stemcells into HSPCs includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL; and

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, and SB-431542 at a concentrationof about 5 μM to form a cell population comprising HSPCs.

In some embodiments, a method for differentiating stem cells into HSPCsincludes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days; and

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, WNT C-59 at a concentration ofabout 2 μM and SB-431542 at a concentration of about 5 μM for 1-3 daysto form a cell population comprising HSPCs.

In some embodiments, an alternative method for differentiating stemcells into HSPCs includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days; and

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL and SB-431542 at a concentration ofabout 5 μM for 1-3 days to form a cell population comprising HSPCs.

In some embodiments, a method for differentiating stem cells into HSPCsincludes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and, optionally, a ROCK inhibitor at aconcentration of about 10 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days; and

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, WNT C-59 at a concentration ofabout 2 μM and SB-431542 at a concentration of about 5 μM for about 2days to form a cell population comprising HSPCs;

In some embodiments, an alternative method for differentiating stemcells into HSPCs includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and, optionally, a ROCK inhibitor at aconcentration of about 10 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days; and

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, and SB-431542 at a concentrationof about 5 μM for about 2 days to form a cell population comprisingHSPCs.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days;

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and an activin/nodalinhibitor for 1-3 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for 1-3 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for up to 8 days; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for at least 6 days and up to 14-28days total to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days;

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L, and an activin/nodal inhibitor for1-3 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for 1-3 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for up to 8 days;

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for up to 6 days; and

(h) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, and SCF for at least 6 days and up to 8-16days total to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days;

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L, and an activin/nodal inhibitor for1-3 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for 1-3 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for up to 8 days;

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for up to 6 days; and

(h) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, SCF and nicotinamide for at least 6 daysand up to 8-16 days total to generate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59 and an activin/nodalinhibitor for about 2 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for about 2 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for 6-8 days; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for at least 6-28 days total togenerate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L and an activin/nodal inhibitor forabout 2 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for about 2 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for 6-8 days; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for 6 day; and

(h) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, SCF and nicotinamide for at least 10-16days to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;

(d) culturing the aggregates in a fourth medium comprising an amount ofFGF2, VEGF, TPO, SCF, IL-3, FLT3L and an activin/nodal inhibitor forabout 2 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for about 2 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for 6-8 days; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for 6 day; and

(h) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, and SCF for at least 10-16 days togenerate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, WNT C-59 at a concentration ofabout 2 μM and SB-431542 at a concentration of about 5 μM to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for a time sufficientto generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, SB-431542 at a concentration ofabout 5 μM to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL;

(h) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, SCF at a concentration of about 20-40 ng/mL and nicotinamide at aconcentration of about 5-10 mM for a time sufficient to generate NKcells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, SB-431542 at a concentration ofabout 5 μM to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL;

(h) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, and SCF at a concentration of about 20-40 ng/mL for a timesufficient to generate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, WNT C-59 at a concentration ofabout 2 μM and SB-431542 at a concentration of about 5 μM for 1-3 daysto form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for 1-3 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for up to 8 days; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for at least 6 daysand up to 21-28 total days to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, SB-431542 at a concentration ofabout 5 μM for 1-3 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for 1-3 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for up to 8 days;

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for up to 6 days; and

(h) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, SCF at a concentration of about 20-40 ng/mL and nicotinamide at aconcentration of about 5-10 mM for at least 6 days and up to 10-16 totaldays to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, SB-431542 at a concentration ofabout 5 μM for 1-3 days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for 1-3 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for up to 8 days;

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for up to 6 days; and

(h) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, and SCF at a concentration of about 20-40 ng/mL for at least 6days and up to 10-16 total days to generate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, WNT C-59 at a concentration ofabout 2 μM and SB-431542 at a concentration of about 5 μM for about 2days to form a cell population comprising HSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for about 2 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for 6-8 days; and

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for 6-28 days togenerate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, and SB-431542 at a concentrationof about 5 μM for about 2 days to form a cell population comprisingHSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for about 2 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for 6-8 days;

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for 6 days; and

(h) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, SCF at a concentration of about 20-40 ng/mL and nicotinamide at aconcentration of about 5-10 mM for 10-16 days to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days;

(d) culturing the aggregates in a fourth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, FLT3L ata concentration of about 10-20 ng/mL, and SB-431542 at a concentrationof about 5 μM for about 2 days to form a cell population comprisingHSPCs;

(e) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for about 2 days;

(f) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for 6-8 days;

(g) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for 6 days; and

(h) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, and SCF at a concentration of about 20-40 ng/mL for 10-16 days togenerate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L to form a cell populationcomprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF; and

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for a time sufficient to generate NKcells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L to form a cell populationcomprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF;

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, SCF and nicotinamide for a time sufficientto generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L to form a cell populationcomprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF;

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, and SCF and for a time sufficient togenerate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for 5-7 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for up to 8 days; and

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for at least 6 days and up to 21-28days total to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for 5-7 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for up to 8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for up to 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, SCF and nicotinamide for at least 6 daysand up to 10-16 days to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 12-24 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for 1-3 days;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for 5-7 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for up to 8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for up to 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, and SCF for at least 6 days and up to10-16 days to generate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for about 6 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for 6-8 days; and

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for at least 6-28 days total togenerate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for about 6 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for 6-8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for about 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, SCF and nicotinamide for at least 10-16days total to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising anamount of a ROCK inhibitor under conditions sufficient to formaggregates for 16-20 hours;

(b) culturing the aggregates in a second medium comprising an amount ofBMP-4, and optionally an amount of a ROCK inhibitor, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising an amount ofBMP-4, FGF2, a WNT pathway activator, and Activin A for about 2 days;

(d) culturing the cell population in a fifth medium comprising an amountof FGF2, VEGF, TPO, SCF, IL-3 and FLT3L for about 6 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3, IL-7, FLT3L, IL-15 and SCF for 6-8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7, FLT3L, IL-15 and SCF for about 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7, FLT3L, IL-15, and SCF for at least 10-16 days total togenerate NK cells

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, to form a cell populationcomprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL; and

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for a time sufficientto generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, to form a cell populationcomprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL;

(f) culturing the cell population in a seventh medium comprising anamount of IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL; and

(g) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, SCF at a concentration of about 20-40 ng/mL, and, nicotinamide ata concentration of about 5-10 mM, for a time sufficient to generate NKcells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM under conditionssufficient to form aggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, to form a cell populationcomprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL;

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL; and

(g) culturing the cell population in an eighth medium comprising anamount IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL and SCF at a concentration of about 20-40 ng/mL for a timesufficient to generate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for 5-7 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for up to 8 days; and

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for at least 6 daysand up to 21-28 total days to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for 5-7 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for up to 8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for up to 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, SCF at a concentration of about 20-40 ng/mL, and nicotinamide ata concentration of about 5-10 mM for at least 6 days and up to 10-16total days to generate NK cells.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 12-48 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 10 μM, for up to 24 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for 1-3 days;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for 5-7 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for up to 8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for up to 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, and SCF at a concentration of about 20-40 ng/mL, for at least 6days and up to 10-16 total days to generate NK cells.

In some embodiments, a method for differentiating stem cells into NKcells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 1 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 6 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for about 6 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for 6-8 days; and

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for 6-28 days togenerate NK cells.

In some embodiments, the HSPCs of step (d) express CD34. In someembodiments, the NK cells express CD56. In some embodiments, the NKcells express at least one activating receptor. In some embodiments, theat least one activating receptor is selected from the group of NKp44,NKp46, CD16, KIR2DL4, and any combination thereof. In some embodiments,the NK cells express at least one inhibitory receptor. In someembodiments, the at least one inhibitory receptor is selected from thegroup of CD94, NKG2A, KIR3DL2, and any combination thereof.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 1 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for about 6 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for 6-8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for about 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, SCF at a concentration of about 20-40 ng/mL, and nicotinamide ata concentration of about 5 to 10 mM for 8 days or 8-16 days to generateNK cells.

In some embodiments, the HSPCs of step (d) express CD34. In someembodiments, the NK cells express CD56. In some embodiments, the NKcells express at least one activating receptor. In some embodiments, theat least one activating receptor is selected from the group of NKp44,NKp46, CD16, KIR2DL4, and any combination thereof. In some embodiments,the NK cells express at least one inhibitory receptor. In someembodiments, the at least one inhibitory receptor is selected from thegroup of CD94, NKG2A, KIR3DL2, and any combination thereof.

In some embodiments, an alternative method for differentiating stemcells into NK cells includes:

(a) culturing a population of stem cells in a first medium comprising aROCK inhibitor at a concentration of about 10 μM for 16-20 hours to formaggregates;

(b) culturing the aggregates in a second medium comprising BMP-4 at aconcentration of about 30 ng/mL, and optionally a ROCK inhibitor at aconcentration of about 1 μM, for 6-10 hours;

(c) culturing the aggregates in a third medium comprising BMP-4 at aconcentration of about 30 ng/mL, FGF2 at a concentration of about 100ng/mL, CHIR-99021 at a concentration of about 7 μM and Activin A at aconcentration of about 2.5-5.0 ng/mL, for about 2 days;

(d) culturing the cell population in a fifth medium comprising FGF2 at aconcentration of about 20 ng/mL, VEGF at a concentration of about 20ng/mL, TPO at a concentration of about 20 ng/mL, SCF at a concentrationof about 100 ng/mL, IL-3 at a concentration of about 40 ng/mL, and FLT3Lat a concentration of about 10-20 ng/mL, for about 6 days to form a cellpopulation comprising HSPCs;

(e) culturing the cell population in a sixth medium comprising an amountof IL-3 at a concentration of about 5 ng/mL, IL-7 at a concentration ofabout 20 ng/mL, FLT3L at a concentration of 10-20 ng/mL, IL-15 at aconcentration of about 10-20 ng/mL and SCF at a concentration of about20 ng/mL, for 6-8 days;

(f) culturing the cell population in a seventh medium comprising anamount IL-7 at a concentration of about 20 ng/mL, FLT3L at aconcentration of 10-20 ng/mL, IL-15 at a concentration of about 10-20ng/mL and SCF at a concentration of about 20 ng/mL for about 6 days; and

(g) culturing the cell population in an eighth medium comprising anamount of IL-7 at a concentration of about 10-20 ng/mL, FLT3L at aconcentration of 5-20 ng/mL, IL-15 at a concentration of about 10-30ng/mL, and SCF at a concentration of about 20-40 ng/mL for 8 days or8-16 days to generate NK cells.

In some embodiments, the HSPCs of step (d) express CD34. In someembodiments, the NK cells express CD56. In some embodiments, the NKcells express at least one activating receptor. In some embodiments, theat least one activating receptor is selected from the group of NKp44,NKp46, CD16, KIR2DL4, and any combination thereof. In some embodiments,the NK cells express at least one inhibitory receptor. In someembodiments, the at least one inhibitory receptor is selected from thegroup of CD94, NKG2A, KIR3DL2, and any combination thereof.

In some embodiments, inducible pluripotent stem cells (iPSC) are thawedand prepared for differentiation culture. Methods for culturing andmaintaining iPSC and other stem cell types are known in the art. In someembodiments, iPSCs are cultured in StemFlex™ Basal Media and StemFlex™Supplement. In some embodiments, prior to inducing differentiation iPSCsare cultured in a medium comprising a low concentration of a ROCKinhibitor (e.g., thiazovivin or Y27632). In some embodiments, iPSCs arecultured in a medium comprising 2 μM of a ROCK inhibitor (e.g.,thiazovivin or Y27632). In some embodiments, after culture in StemFlex™Basal Media and StemFlex™ Supplement cells are resuspended in the mediain Table 18A or 18B. In some embodiments, cells are cultured in themedia in Table 18A or 18B for 16 to 20 hours. In some embodiments, cellsare removed from the Table 18A or 18B media and are resuspended in theTable 19A or 19B media and cultured for about 8 hours. In someembodiments, after culturing in the media in Table 19A or 19B afterabout 8 hours, the media is diluted in half by the addition of the mediain Table 20A or 20B, cells are then cultured for about 48 hours. In someembodiments, after culture for 48 hours in the media in Table 20A or 20Bcells are transferred to the media in Table 21A or 21B and cultured forabout 48 hours. In some embodiments, after culture for about 48 hours inthe media in Table 21A or 21B, cells are transferred to the media inTable 22 and cultured for about 48 hours. In some embodiments, afterculture for about 48 hours in the media in Table 22, cells aretransferred to the medium in Table 23A or 23B and cultured for about 4days. In some embodiments, after culture for about 4 days in the mediain Table 23A or 23B, half of the media is replaced with fresh media fromTable 23A or 23B and cells are cultured for an additional 4 days. Insome embodiments, after culture for about 4 days in the media in Table23A or 23B, cells are transferred to the media in Table 24A or 24B arecultured for about 3 days and a full media change occurs every 2-3 daysfor up to 28 days. In some embodiments, NK cells are formed duringculture with media in Table 24A or 24B. In some embodiments, afterculturing in the media in Tables 23A or 23B, cells are transferred tothe media in Table 24A or 24B and are cultured for up to 6 days followedby a full media change to the media in Table 25A or Table 25B, wherein afull media change occurs every 2-3 days for up to 10-16 days. In someembodiments, a partial media change is performed with the media in Table25A or Table 25B at a time during the 10-16 days duration. In someembodiments, NK cells are formed during culture with media in Table 25Aor Table 25B.

In some embodiments, iPSCs are thawed and prepared for differentiation,in StemFlex™ Basal Media and StemFlex™ Supplement, then cells arecultured sequentially in the following order a) following culture inStemFlex™ Basal Media and StemFlex™ Supplement cells are resuspended andcultured in in the media in Table 18A for 16 to 20 hours; b) cells areremoved from the Table 18A media and are resuspended in the Table 19media and cultured for about 8 hours; c) after culturing in the media inTable 19A or 19B for about 8 hours, the media is diluted in half by theaddition of the media in Table 20A, cells are then cultured for about 48hours; d) after culture for about 48 hours in the media in Table 20Acells are transferred to the media in Table 21A and cultured for about48 hours; e) after culture for about 48 hours in the media in Table 21A,cells are transferred to the media in Table 22 and cultured for about 48hours; f) after culture for about 48 hours in the media in Table 22,cells are transferred to the medium in Table 23A and cultured for about4 days; g) after culture for about 4 days in the media in Table 23A,half of the media is replaced with fresh media from Table 23A and cellsare cultured for an additional 4 days; h) after culture for about 4 daysin the media in Table 23A, cells are transferred to the media in Table24A are cultured for about 3 days and a full media change occurs every2-3 days for up to 28 days; i) NK cells are formed.

In some embodiments, iPSCs are thawed and prepared for differentiation,in StemBrew™ Basal Media and StemBrew™ Supplement, then cells arecultured sequentially in the following order a) following culture inStemBrew™ Basal Media and StemBrew™ Supplement cells are resuspended andcultured in in the media in Table 18B for 16 to 20 hours; b) cells areremoved from the Table 18B media and are resuspended in the Table 19A or19B media and cultured for about 8 hours; c) after culturing in themedia in Table 19 for about 8 hours, the media is diluted in half by theaddition of the media in Table 20B, cells are then cultured for about 48hours; d) after culture for about 48 hours in the media in Table 20Bcells are transferred to the media in Table 21B and cultured for about48 hours; e) after culture for about 48 hours in the media in Table 21B,cells are transferred to the media in Table 22 and cultured for about 48hours; f) after culture for about 48 hours in the media in Table 22,cells are transferred to the medium in Table 23B and cultured for about4 days; g) after culture for about 4 days in the media in Table 23B, themedia is replaced with fresh media from Table 23B and cells are culturedfor an additional 4 days; h) after culture for about 4 days in the mediain Table 23B, cells are transferred to the media in Table 24B arecultured for about 6 days, with fresh media from Table 24B changed after3 days; i) after culture for about 6 days in the media in Table 24Bcells are transferred to the media in Table 25A or Table 25B and a fullmedia change occurs every 2-3 days for up to 16 days; i) NK cells areformed.

In some embodiments, cells are cultured in aggregates. In someembodiments, cells are cultured in aggregates until day 5 ofdifferentiation. In some embodiments, aggregates are present in theculture as late as day 20 of differentiation. In some embodiments,single cells emerge during differentiation. In some embodiments, singlecells emerge on day 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 of differentiation. In some embodiments, cell aggregatesdissociate into single cells during culture. In some embodiments,aggregates dissociate into single cells on any one of days 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

In some embodiments, aggregates are about 50 μm to about 200 μm indiameter. In some embodiments, aggregates are about 100 μm to about 200μm in diameter. In some embodiments, aggregates are less than about 100μm in diameter. In some embodiments, aggregates are about 50 μm to about100 μm in diameter. In some embodiments, aggregates are about 60 μm toabout 100 μm in diameter.

In some embodiments, at least 99%, at least 90%, at least 80%, at least70%, at least 60%, at least 50%, at least 40%, at least 30%, at least20%, at least 10%, at least 5%, or at least 1% of the cells in cultureare in aggregates. In some embodiments, at least 99%, at least 90%, atleast 80%, at least 70%, at least 60%, at least 50%, at least 40%, atleast 30%, at least 20%, at least 10%, at least 5%, or at least 1% ofthe cells in culture are single cells. Differentiating Cell Phenotypes

Throughout differentiation from stem cell to natural killer cell, or anyintermediate cell types therein, cells express a variety of phenotypicmarkers. Similarly, differentiation from stem cell to HSPC or HSPC to NKcell provide one or more markers of cell types during differentiation.In some embodiments, at day 0 of differentiation, cells are Oct3/4+ andSox2+. In some embodiments, cells are Oct3/4⁺ and Sox2⁺ on any one ofday 1, 2, 3, or 4 of differentiation. In some embodiments, cells loseOct3/4 and Sox2 expression beginning at day 2. In some embodiments,cells express CD34 beginning at day 3. In some embodiments, at any oneor more of day 4, 5, 6, 7 or 8 of differentiation, HSCs areCD34⁺/CD43⁺/CD45⁻. In some embodiments, at day 6 of differentiation,HSCs are CD34⁺/CD43⁺/CD45⁻. In some embodiments, at one or more of day10, 11, 12, 13, 14, cells are CD34⁺/CD43⁺/CD45⁺. In some embodiments, atany one or more of days 12, 13, 14, 15, or 16, CLPs areCD34⁻/CD45⁺/CD38⁺/CD117⁺/CD7⁺. In some embodiments, at day 14 ofdifferentiation, CLPs are CD34-/CD45⁺/CD38⁺/CD117⁺/CD7⁺. In someembodiments, on any one or more of days 17, 18, 19, 20, 21, or 22 ofdifferentiation, immature NK cells areCD34⁻/CD45⁺/CD56⁺/NKp46⁺/CD94⁺/NKG2A⁺. In some embodiments, on day 20 ofdifferentiation, immature NK cells areCD34⁻/CD45⁺/CD56⁺/NKp46⁺/CD94⁺/NKG2A⁺. In some embodiments on any one ormore of days 26, 27, 28, 29, or 30 of differentiation, NK cells areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16^(−/+)/and KIR⁺. Insome embodiments, NK cells formed in step (c) or step (d) of Stage IIare CD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁺/KIR⁺. In someembodiments, NK cells formed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁻/KIR⁻. In someembodiments, NK cells formed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁺/KIR. In someembodiments, NK cells formed in step (c) or step (d) of Stage II areCD45⁺/CD56⁺/NKp44⁺/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16⁻/KIR⁺. In someembodiments on day 28 of differentiation, NK cells areCD45⁺/CD56⁺/NKp44*/NKp46⁺/CD94⁺/NKG2A⁺/NKG2D⁺/CD16^(−/+)/KIR^(−/+). Insome embodiments, the differentiated NK cells do not express CD3. Insome embodiments, on days 21 and 35, differentiated NK cells do notexpress CD3.

In some embodiments, on day 6 at least about 38-55% of the cells areCD34⁺. In some embodiments, at least about 29/6-49% of cells areCD34⁺/CD45⁺. In some embodiments, at day 14, at least about 19-45% ofcells are CD7⁺/CD45⁺. In some embodiments, at day 17 about 50% of cellsare CD56⁺/CD45⁺. In some embodiments, at day 20, at least about 70% ofcells are CD56⁺/CD45⁺.

In some embodiments, about 20-60% of differentiating cells areCD34⁺/CD43⁻ on day 6 of differentiation. In some embodiments, 0 to about5% of differentiating cells are CD34⁺/CD43⁺ on day 6 of differentiation.In some embodiments, about 15% to about 25% of differentiating cells areCD34⁺/CD43⁺ on day 10 of differentiation. In some embodiments, about 45%to about 55% of differentiating cells are CD34⁺/CD43⁻ on day 10 ofdifferentiation.

In some embodiments, about 50% to about 60% of differentiating cells areCD45⁺/CD56⁻ on day 10 of differentiation. In some embodiments, about 50%to about 75% of differentiating cells are CD45⁺/CD56⁻ on day 14 ofdifferentiation. In some embodiments, about 5% to about 40% ofdifferentiating cells are CD45⁺/CD56⁺ on day 14 of differentiation. Insome embodiments, about 50% of differentiating cells are CD45⁺/CD56⁺ onday 17 of differentiation. In some embodiments, about 20% to about 30%of differentiating cells are CD45⁺/CD56⁻ on day 20 of differentiation.In some embodiments, about 65% to about 90% of differentiating cells areCD45⁺/CD56⁺ on day 20 of differentiation. In some embodiments, about 95%to 100% of differentiating cells are CD45⁺/CD56⁺ on day 28 ofdifferentiation. In some embodiments, about 95% to 100% ofdifferentiating cells are CD45⁺/CD56⁺ on day 35 of differentiation.

In some embodiments, differentiating cells lose Sox2 expression by day1, 2, 3, or 4 of differentiation. In some embodiments, differentiatingcells lose Oct3/4 expression by day 1, 2, 3, or 4 of differentiation. Insome embodiments, by day 6 of differentiation, cells do not expressOCT3/4. In some embodiments, by day 6 of differentiation, cells do noexpress Sox2.

In some embodiments, about 35% to about 45% of differentiating cellsexpress NKp44 on day 21 of differentiation. In some embodiments, about70% to about 80% of differentiating cells express NKp44 on day 28 ofdifferentiation. In some embodiments, about 75% to about 85% ofdifferentiation cells express NKp44 on day 37 of differentiation. Insome embodiments, about 60% to about 70% of differentiating cellsexpress NKp46 on day 21 of differentiation. In some embodiments, about75% to about 85% of differentiating cells express NKp46 on day 28 ofdifferentiation. In some embodiments, about 70% to about 80% ofdifferentiation cells express NKp46 on day 37 of differentiation. Insome embodiments, about 15% to about 30% of differentiating cellsexpress CD16 on day 21 of differentiation. In some embodiments, about10% to about 30% of differentiating cells express CD16 on day 28 ofdifferentiation. In some embodiments, about 1% to about 10% ofdifferentiation cells express CD16 on day 37 of differentiation. In someembodiments, differentiating cells do not express KIR2DL4 on day 21 ofdifferentiation. In some embodiments, about 2% to about 5% ofdifferentiating cells differentiating cells express KIR2DL4 on day 28 ofdifferentiation. In some embodiments, about 20% to about 40% ofdifferentiating cells express KIR2DL4 on day 37 of differentiation. Insome embodiments, about 75% to about 85% of differentiating cellsexpress CD94 on day 21 of differentiation. In some embodiments, about80% to about 90% of differentiating cells express CD94 on day 28 ofdifferentiation. In some embodiments, about 65% to about 80% ofdifferentiation cells express CD94 on day 37 of differentiation. In someembodiments, about 70% to about 80% of differentiating cells expressNKG2A on day 21 of differentiation. In some embodiments, about 75% toabout 85% of differentiating cells express NKG2A on day 28 ofdifferentiation. In some embodiments, about 70% to about 80% ofdifferentiation cells express NKG2A on day 37 of differentiation. Insome embodiments, differentiating cells do no express KIR3DL2 on day 21of differentiation. In some embodiments, differentiating cells do notexpress KIR3DL2 on day 28 of differentiation. In some embodiments, 0% toabout 5% of differentiation cells express KIR3DL2 on day 37 ofdifferentiation.

In some embodiments, on day 20 of differentiation, cells have an about10 to about 100-fold change increase normalized to day 0 of one or moreof EOMES, NFIL3, FCGR3A, KIR2DL1, KIR2DS4, KIR2DL3, KIR3DL1, KIR3DL2,IL15, IL18, IL2RA, KLRF1 (NKP80), SLAMF7. In some embodiments, on day 35of differentiation, cells have about 10 to about 100-fold changeincrease normalized to ay 0 of one or more of EOMES, NFIL3, FCGR3A,GZMM, IL15, KLRF1 (NKP80), KLRD1 (CD94).

In some embodiments, on day 20 of differentiation, cells have about 100to about 1000-fold change increase normalize to day 0 of one or more ofTBX21, NCR1, NCR2, CCR5, CD226 (DNAM-1), GZMM, IL2RB, KLRD1 (CD94). Insome embodiments, on day 35 of differentiation, cells have about 100 toabout 1000-fold change increase normalized to day 0 of one or more ofTBX21, NCR1, NCR2, KIR2DL1, KIR3DL1, KIR3DL2, IL2RA, IL2RB, SLAMF7.

In some embodiments, on day 20 of differentiation, cells have greaterthan 1000-fold change increase normalized to day 0 of one or more ofGZMA, GZMH, GZMK, NCR3, CCL3, CCL4, CCL5, CCR1, IL2RG, KLRB1, KLRC1(NKG2A), KLRC2 (NKG2C). In some embodiments, on day 35 ofdifferentiation, cells have great than a 1000-fold change increasecompared to day 0 of one or more of GZMA, GZMH, GZMK, NCR3, CCL3, CCL4,CCL5, CCR1, CCR5, CD226 (DNAM-1), IL2RG, KIR2DL1, KIR2DS4, KLRB1, KLRC1(NKG2A), KLRC2 (NKG2C).

In some embodiments, about 25% to about 35% of differentiating cellsexpress Granzyme B on day 16 of differentiation. In some embodimentsabout 25% to about 35% of differentiating cells express Perforin on day16 of differentiation. In some embodiments, about 90% to about 95% ofdifferentiating cells express Granzyme B on day 24 of differentiation.In some embodiments, about 75% to about 80% of differentiating cellsexpress Perforin on day 24 of differentiation.

In some embodiments, differentiating cells in media with GSK3b blockersincreases CD34 expression. In some embodiments, differentiating cells inmedia with CHIR-99021 and/or Activin A enhances CD34 expression.

In some embodiments, NK cells are CD45⁺/CD56⁺. In some embodiments, onday 17 of differentiation the culture comprises CD45⁺/CD56⁺ cells.

Differentiated Natural Killer Cells

Natural killer (NK) cells are a subpopulation of lymphocytes which playa critical role in the innate immune system. NK cells have cytotoxicityagainst a variety of cells including but not limited to tumor cells andvirus-infected cells. In some embodiments, the stem cells describedherein are differentiated to NK cells. In some embodiments, iPSCs aredifferentiated into NK cells. In some embodiments HSPCs aredifferentiated into NK cells.

NK cells have been shown to secrete cytokines. In some embodiments, anNK cell differentiated or obtained by using the methods described hereinsecretes at least one cytokine to the same or similar levels as anendogenous NK cell. In some embodiments, an NK cell differentiated orobtained by using the methods described herein secretes at least onecytokine to the same or similar levels as a wild-type NK cell line (e.g.NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, and IMC-1). In some embodiments,an NK cell differentiated or obtained by using the methods describedherein secretes at least one cytokine to the same or similar levels asan NK cell derived using any differentiation method known to those ofskill in the art. In some embodiments, an NK cell differentiated orobtained by using the methods described herein secretes at least onecytokine to the same or similar levels as an NK cell derived from humanembryonic stem cells (hESCs), peripheral blood (PB-NK), umbilical cordblood (UCB-NK), or bone marrow (BM-NK). In some embodiments, an NK celldifferentiated or obtained by using the methods described hereinsecretes at least one cytokine to the same or similar levels as a tissueresident or tumor infiltrating NK cell. In some embodiments, the NKcells differentiated or obtained by using the methods described hereinsecrete TNF-α, IL-10, IFN-γ, GM-CSF, TGF-β IL-5, IL13, or anycombination thereof.

NK cells have been shown to secrete chemokines. In some embodiments, anNK cell differentiated or obtained by using the methods described hereinsecretes at least one chemokine to the same or similar levels as anendogenous NK cell. In some embodiments, an NK cell differentiated orobtained by using the methods described herein secretes at least onechemokine to the same or similar levels as a wild-type NK cell line(e.g. NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, and IMC-1). In someembodiments, an NK cell differentiated or obtained by using the methodsdescribed herein secretes at least one chemokine to the same or similarlevels as an NK cell derived using any differentiation method known tothose of skill in the art. In some embodiments, an NK celldifferentiated or obtained by using the methods described hereinsecretes at least one chemokine to the same or similar levels as an NKcell derived from human embryonic stem cells (hESCs), peripheral blood(PB-NK), umbilical cord blood (UCB-NK), or bone marrow (BM-NK). In someembodiments, an NK cell differentiated or obtained by using the methodsdescribed herein secretes at least one chemokine to the same or similarlevels as a tissue resident or tumor infiltrating NK cell. In someembodiments, the NK cells differentiated or obtained by using themethods described herein secrete CCL1, CCL2, CCL3, CCL4, CCL5, CXCL8,MIP-1α, MIP-1β, IL-8, RANTES, or any combination thereof. Methods ofmeasuring cytokines and chemokines are known to those of skill in theart. Examples of methods include but are not limited to quantitativepolymerase chain reaction (q-PCR), enzyme-linked immunosorbent assay(ELISA), or flow cytometry analysis.

NK cells have both activating and inhibitory receptors that regulatetheir function. In some embodiments, an NK cell differentiated orobtained by using the methods described herein expresses at least oneactivating receptor similar to, or at the same level as an endogenous NKcell, a wild-type NK cell line, an NK cell derived from a sample ortissue as described herein, and/or an NK cell derived from adifferentiation method known to one of skill in the art. In someembodiments, the NK cells differentiated or obtained by using themethods described herein express any one or more of the activatingreceptors: NKG2D, NKG2C, Ly49D, Ly49H, KIR2DL4, KIR2DS1, KIR2DS2,KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKp30, NKp46, NKp44, NKR-P1C,NKR-P1F, NKR-P1G, or DNAM-1. In some embodiments, a function of adifferentiated NK cell associated with at least one activating receptoris induced upon stimulation of the activating receptor at the same orsimilar level as an endogenous NK cell, a wild-type NK cell line, an NKcell derived from a sample or tissue as described herein, and/or an NKcell derived from a differentiation method known to one of skill in theart. In some embodiments, an NK cell differentiated or obtained by usingthe methods described herein expresses at least one inhibitory receptorsimilar to, or at the same level as an endogenous NK cell, a wild-typeNK cell line, an NK cell derived from a sample or tissue as describedherein, and/or an NK cell derived from a differentiation method known toone of skill in the art. In some embodiments, the NK cellsdifferentiated or obtained by using the methods described herein expressany one or more of the inhibitory receptors: Ly49A, Ly49C, Ly49I, Ly49P,KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKR-PIA, NKR-P1B, NKR-P1D,NKG2A, ILT2, or CD244. In some embodiments, a function of adifferentiated NK cell associated with at least one inhibitory receptoris induced upon stimulation of the activating receptor at the same orsimilar level as an endogenous NK cell, a wild-type NK cell line, an NKcell derived from a sample or tissue as described herein, and/or an NKcell derived from a differentiation method known to one of skill in theart.

In some embodiments, the differentiated NK cells express at least one,two, three, four, five, six, seven, eight or all of the followingmarkers: CD45, CD56, CD94, NKG2A, CD16, NKp44, NKp46, KIR2DL4, andKIR3DL2. In some embodiments, the differentiated NK cells express atleast one, two, three, four, five or all of the following markers: CD56,NKp44, NKp46, CD94, NKG2A and KIR2DL4. In some embodiments, thedifferentiated NK cells have at least 25%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or at least 99% of the cell population expressing one, two,three, four, five, six, seven, eight or all of the following markers:CD45, CD56, CD94, NKG2A, CD16, NKp44, NKp46, KIR2DL4, and KIR3DL2. Insome embodiments, the differentiated NK cells have at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or at least 99% of the cellpopulation expressing one, two, three, four, five or all of thefollowing markers: CD56, NKp44, NKp46, CD94, NKG2A and KIR2DL4.

In some embodiments, the differentiated NK cells express at least one,two, three or all of the following markers: CD38, CD96, DNAM-1, andICAM-1. In some embodiments, the differentiated NK cells express atleast one, two, three or all of the following markers: CD38, CD96,DNAM-1, and ICAM-1. In some embodiments, the differentiated NK cellshave at least 25%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% or at least99% of the cell population expressing one, two, three or all of thefollowing markers: CD38, CD96, DNAM-1, and ICAM-1. In some embodiments,the differentiated NK cells have at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95% or at least 99% of the cell population expressing one,two, three or all of the following markers: CD38, CD96, DNAM-1, andICAM-1.

In some embodiments, the differentiated NK cells express at least one,two, three or all of the following markers: NKG2D, TIM3, CD16, and CD25.In some embodiments, the differentiated NK cells express at least one,two, three or all of the following markers: NKG2D, TIM3, CD16, and CD25.In some embodiments, the differentiated NK cells have at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or at least 99% of the cell population expressing one, two,three or all of the following markers: NKG2D, TIM3, CD16, and CD25. Insome embodiments, the differentiated NK cells have at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or at least 99% of the cell population expressing one, two,three or all of the following markers: NKG2D, TIM3, CD16, and CD25.

In some embodiments, the NK cells differentiated herein have the same orsimilar functional capacity to that of endogenous NK cells, a wild-typeNK cell line, an NK cell derived from a sample or tissue as describedherein, and/or an NK cell derived from a differentiation method known toone of skill in the art.

In some embodiments, the differentiated NK cells are capable of beingactivated. In some embodiments, the differentiated NK cells are capableof being activated to the same or similar level as endogenous NK cells,a wild-type NK cell line, an NK cell derived from a sample or tissue asdescribed herein, and/or an NK cell derived from a differentiationmethod known to one of skill in the art. Activation refers to NK cellsthat have received an activating signal and have the potential forkilling target cells. NK cell cytotoxicity and activation contribute totheir anti-tumor activity. In some embodiments, the NK cells have thesame or similar anti-tumor activity as an endogenous NK cell, awild-type NK cell line, an NK cell derived from a sample or tissue asdescribed herein, and/or an NK cell derived from a differentiationmethod known to one of skill in the art.

The cytotoxic response of NK cells involves a process known asdegranulation. Degranulation leads to the release of cytotoxic moleculessuch as perforin and granzyme which mediate apoptosis of target cells.An additional maker of NK cell degranulation is CD107a. Followingstimulation of NK cells after contact with MHC devoid targets, CD107a isupregulated on the cell surface. In some embodiments, the differentiatedNK cells express CD107a to the same or similar levels as endogenous NKcells, a wild-type NK cell line, an NK cell derived from a sample ortissue as described herein, and/or an NK cell derived from adifferentiation method known to one of skill in the art. Measurement ofPerforin, Granzyme, and CD107a are used in the art as a readout ofcytotoxic activity. Methods of measuring Perforin, Granzyme, and CD107aare known in the art and include but are not limited to, flow cytometricdetection, colorimetric assays (see e.g., Hagn et al. 2014 J Vis. Exp.93:52419), and Western blot analysis.

Additional methods to measure the cytotoxicity of NK cells include, butare not limited to, in vitro analysis of effector:target cell killing.Target cell death is measured using methods including culturing thechromium release assay, apoptosis assays, or counting total cell number.In some embodiments, the NK cells have the same or similar cytotoxicityas endogenous NK cells, a wild-type NK cell line, an NK cell derivedfrom a sample or tissue as described herein, and/or an NK cell derivedfrom a differentiation method known to one of skill in the art. In someembodiments, the NK cells kill target cells at the same or similar rateas endogenous NK cells, a wild-type NK cell line, an NK cell derivedfrom a sample or tissue as described herein, and/or an NK cell derivedfrom a differentiation method known to one of skill in the art.

In any of the embodiments provided herein, the target cells may includetumor cells (e.g., myelogenous leukemia cells, lymphoma cells, melanomacells, or other metastatic tumor cells. In some embodiments, the targetcells may comprise immortalized tumor cells such as immortalizedmyelogenous leukemia cells (e.g., K-562 cells), or immortalized Hodgkinlymphoma cells (e.g., L-428, L-540, or KM-H2 cells). In someembodiments, the target cells may comprise tumor cells from chroniclymphocytic leukemia (CLL), non-Hodgkin lymphomas (e.g., diffuse largeB-cell lymphoma (DLBCL), high grade B-cell lymphoma, transformedfollicular lymphoma (FL), grade 3B FL, and Richter's transformation ofCLL), and acute lymphoblastic leukemia (ALL). In some embodiments, thetarget cells may comprise tumor cells from Hodgkin lymphoma. In variousembodiments, the target cells may be cultured in vitro.

In some embodiments, the differentiated NK cells use antibody-dependentcytotoxicity (ADCC) at the same or similar levels to endogenous NKcells, a wild-type NK cell line, an NK cell derived from a sample ortissue as described herein, and/or an NK cell derived from adifferentiation method known to one of skill in the art. NK cellsexpress Fcγ receptors on their surface that are activated by binding toantibodies on target cells. Receptor activation activates the release ofcytokines from the NK cell. In some embodiments, the NK cells have thesame, or similar ADCC to that of endogenous NK cells, a wild-type NKcell line, an NK cell derived from a sample or tissue as describedherein, and/or an NK cell derived from a differentiation method known toone of skill in the art. Assays to measure ADCC are known in the art andinvolved culturing NK cells with a target cell. Binding of the receptorto an antibody on the target cell initiates ADCC which is measured bychromium release assay.

In some embodiments, the differentiated NK cells proliferate at the samerate as endogenous NK cells, a wild-type NK cell line, an NK cellderived from a sample or tissue as described herein, and/or an NK cellderived from a differentiation method known to one of skill in the art.Proliferation is measured using methods known in the art. Methodsinclude but are not limited to manual counting of cell numbers,measurement of proliferation markers such as Ki67, DNA labeling and cellcycle analysis.

In some embodiments, the differentiated NK cells do not exhibitexhaustion or exhibit a low level of exhaustion (e.g., a level ofexhaustion markers associated with a functional NK cell). In someembodiments, exhaustion is detected by detecting a reduced expression ofIFNγ, granzyme B, perforin, CD107a, and/or TNFα in cells. In someembodiments, exhaustion is detected by detecting increased expression(e.g., on the surface of the cell) of an exhaustion marker, e.g., PD-1,LAG-3, TIGIT and/or TIM-3.

In some embodiments, the differentiated NK cells persist in vitro or invivo In some embodiments, persistence of the cells is assessed byanalyzing their presence and quantity in one or more tissue samples thatare collected from a subject following administration of the cells tothe subject. In some embodiments, persistence is defined as the longestduration of time from administration to a time wherein a detectablelevel of the cells is present in a given tissue type (e.g., peripheralblood). In some embodiments, persistence is defined as the continuedabsence of disease (e.g., complete response or partial response).Determination of the absence of disease and response to treatment areknown to those of skill in the art and described herein.

Genome Editing

In some embodiments, a cell described herein comprises at least one geneedit and is referred to as an “engineered cell.” In some embodiments, astem or progenitor cell comprises at least one gene edit, and that editis maintained through differentiation. In some embodiments, a stem orprogenitor cell comprises at least one gene edit, and that edit ismaintained through differentiation into an NK cell such that the NK cellcomprises the gene edit.

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner. In some embodiments, genome editing methods,e.g., the CRISPR-endonuclease system, are used to genetically modify acell as described herein, e.g., to create a gene-edited iPSC cell. Insome embodiments, genome editing methods e.g., the CRISPR-endonucleasesystem, are used to genetically modify a cell as described herein. Anygenome editing method to known to one of skill in the art is useful forengineering the cells described herein.

Examples of methods of genome editing described herein include methodsof using site-directed nucleases to cut deoxyribonucleic acid (DNA) atprecise target locations in the genome, thereby creating single-strandor double-strand DNA breaks at particular locations within the genome.Such breaks can be and regularly are repaired by natural, endogenouscellular processes, such as homology-directed repair (HDR) andnon-homologous end joining (NHEJ), as described in Cox et al.,“Therapeutic genome editing: prospects and challenges,”, NatureMedicine, 2015, 21(2), 121-31. These two main DNA repair processesconsist of a family of alternative pathways. NHEJ directly joins the DNAends resulting from a double-strand break, sometimes with the loss oraddition of nucleotide sequence, which may disrupt or enhance geneexpression. HDR utilizes a homologous sequence, or donor sequence, as atemplate for inserting a defined DNA sequence at the break point. Thehomologous sequence can be in the endogenous genome, such as a sisterchromatid. Alternatively, the donor sequence can be an exogenouspolynucleotide, such as a plasmid, a single-strand oligonucleotide, adouble-stranded oligonucleotide, a duplex oligonucleotide or a virus,that has regions (e.g., left and right homology arms) of high homologywith the nuclease-cleaved locus, but which can also contain additionalsequence or sequence changes including deletions that can beincorporated into the cleaved target locus. A third repair mechanism canbe microhomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ,” in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJcan make use of homologous sequences of a few base pairs flanking theDNA break site to drive a more favored DNA end joining repair outcome,and recent reports have further elucidated the molecular mechanism ofthis process; see, e.g., Cho and Greenberg, Nature, 2015, 518, 174-76;Kent et al., Nature Structural and Molecular Biology, 2015, 22(3):230-7;Mateos-Gomez et al., Nature, 2015, 518, 254-57; Ceccaldi et al., Nature,2015, 528, 258-62. In some instances, it may be possible to predictlikely repair outcomes based on analysis of potential microhomologies atthe site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenetic modifications. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of endonucleases.

Exemplary Gene Edits

In some embodiments, an engineered cell (e.g., an NK cell derived from agene-edited iPSC) evades an immune response and/or survives followingengraftment into a subject at higher success rates than an unmodifiedcell. In some embodiments, an engineered cell is hypoimmunogenic. Insome embodiments, an engineered cell has improved (i) persistency; (ii)immune evasiveness; (iii) cytotoxic activity; (iv) ADCC activity; and/or(v) anti-tumor activity, as compared to an unmodified or wild-type cell.

In certain embodiments, any cells described herein can be gene-editedusing any of the gene-editing methods. In some embodiments, a disruptedgene is a gene that does not encode functional protein. In someembodiments, a cell that comprises a disrupted gene does not express(e.g., at the cell surface) a detectable level (e.g. by antibody, e.g.,by flow cytometry) of the protein encoded by the gene. A cell that doesnot express a detectable level of the protein may be referred to as aknockout cell.

In some embodiments, the cells described herein are gene-edited todisrupt one or more of the genes encoding an MHC-I or MHC-II humanleukocyte antigen, a component of a MHC-I or MHC-II complex, or atranscriptional regulator of a MHC-I or MHC-II complex. In someembodiments, the cells described herein are gene-edited to disrupt oneor more of the genes encoding an MHC-I or MHC-II human leukocyteantigen. In some embodiments, the cells described herein are gene-editedto disrupt one or more of the genes encoding one or more components of aMHC-I or MHC-II complex. In some embodiments, the cells described hereinare gene-edited to disrupt one or more of the genes encoding one or moretranscriptional regulator of a MHC-I or MHC-II complex.

In some embodiments, the cells described herein are gene-edited todisrupt one or more genes including but not limited to:beta-2-microglobulin (B2M), class II major histocompatibility complextransactivator (CIITA), ADAM metallopeptidase domain 17 (ADAM17),cytokine inducible SH2 containing protein (CISH), Regnasel, Fas cellsurface death receptor (FAS), T cell immunoreceptor with Ig and ITIMdomains (TIGIT), programmed cell death 1 (PD-1), NKG2-A type II integralmembrane protein-like (NKG2A), CD70, aurora like protein kinase 4(ALK4), and/or type I activin receptor (e.g., conditionally). In someembodiments, the cells described herein are gene-edited to improveimmune evasiveness. In some embodiments, immune evasiveness is improvedby disrupting a gene encoding a polypeptide associated with inducing animmune response. In some embodiments, immune evasiveness is improved bydisrupting B2M and/or CIITA gene(s).

In some embodiments, the cells described herein are gene-edited toimprove persistence of the cell or differentiated cell. In someembodiments, persistence is improved by gene-editing a cell to insert apolynucleotide encoding, without limitation, one or more of thefollowing: interleukin 15 (IL15), interleukin 15 receptor, alpha chain(IL15Ra), Serpin Family B Member 9 (SERPINB9), and class Ihistocompatibility antigen, alpha chain E (HLA-E).

In some embodiments, the cells described herein are gene-edited toimprove antibody-dependent cellular cytotoxicity (ADCC) of the cell ordifferentiated cell. Natural Killer cells express Fcγ receptors on theirsurface which bind to target cells by recognizing antibodies (e.g. IgG)on the surface. The binding induces a signal cascade resulting incytokine production and cytotoxic activation of the NK cell (ADCC).Increased ADCC contributes to the anti-tumor activity of an NK cell. Oneexample of the Fcγ receptors is CD16. Increased expression of CD16 onthe surface or NK cells increases their ADCC potential. In someembodiments, the cells described herein are gene-edited to insert apolynucleotide encoding CD16 (e.g., a high affinity non-cleavable CD16).

In some embodiments, the cells described herein are gene-edited toinsert a polynucleotide encoding one or more chimeric antigen receptors(CARs). CARs are designed to enhance a cells ability to recognize, bindto, and kill tumor cells. In some embodiments, the CAR enhances the NKcells ability to recognize tumor cells. In some embodiments, the CARenhances the NK cells anti-tumor activity. In some embodiments, andwithout limitation, the CAR is a TNF receptor superfamily member 17(BCMA) CAR, CD30 CAR, CD19 CAR, CD33 CAR, NKG2D CAR (or a CAR orreceptor comprising an NKG2D ectodomain), CAR CD70 CAR, NKp30 CAR, CD73CAR, G protein coupled receptor 87 (GPR87) CAR and solute carrier family7 member 11 (SLC7A11 (xCT)) CAR.

CRISPR Endonuclease System

The CRISPR-endonuclease system is a naturally occurring defensemechanism in prokaryotes that has been repurposed as a RNA-guidedDNA-targeting platform used for gene editing. Accordingly, in someembodiments, a CRISPR-endonuclease system is utilized to introduce agene edit into a cell. CRISPR systems include Types I, II, III, IV, V,and VI systems. In some embodiments, the CRISPR system is a Type IICRISPR/Cas9 system. In some embodiments, the CRISPR system is a Type VCRISPR/Cprf system. CRISPR systems rely on a DNA endonuclease, e.g.,Cas9, and two noncoding RNAs—crisprRNA (crRNA) and trans-activating RNA(tracrRNA)—to target the cleavage of DNA.

The crRNA drives sequence recognition and specificity of theCRISPR-endonuclease complex through Watson-Crick base pairing, typicallywith a ˜20 nucleotide (nt) sequence in the target DNA. Changing thesequence of the 5′ 20 nt in the crRNA allows targeting of theCRISPR-endonuclease complex to specific loci. The CRISPR-endonucleasecomplex only binds DNA sequences that contain a sequence match to thefirst 20 nt of the single-guide RNA (sgRNA) if the target sequence isfollowed by a specific short DNA motif (with the sequence NGG) referredto as a protospacer adjacent motif (PAM).

TracrRNA hybridizes with the 3′ end of crRNA to form an RNA-duplexstructure that is bound by the endonuclease to form the catalyticallyactive CRISPR-endonuclease complex, which can then cleave the targetDNA.

Once the CRISPR-endonuclease complex is bound to DNA at a target site,two independent nuclease domains within the endonuclease each cleave oneof the DNA strands three bases upstream of the PAM site, leaving adouble-strand break (DSB) where both strands of the DNA terminate in abase pair (a blunt end).

In some embodiments, the endonuclease is a Cas9 (CRISPR associatedprotein 9). In some embodiments, the Cas9 endonuclease is fromStreptococcus pyogenes, although other Cas9 homologs may be used, e.g.,S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR 1 Cas9, S.thermophilus CRISPR 3 Cas9, or T. denticola Cas9. In some embodiments,the CRISPR endonuclease is Cpf1, e.g., L. bacterium ND2006 Cpf1 orAcidaminococcus sp. BV3L6 Cpf1. In some embodiments, the endonuclease isCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease. In some embodiments,wild-type variants may be used. In some embodiments, modified versions(e.g., a homolog thereof, a recombination of the naturally occurringmolecule thereof, codon-optimized thereof, or modified versions thereof)of the preceding endonucleases may be used.

The CRISPR nuclease can be linked to at least one nuclear localizationsignal (NLS). The at least one NLS can be located at or within 50 aminoacids of the amino-terminus of the CRISPR nuclease and/or at least oneNLS can be located at or within 50 amino acids of the carboxy-terminusof the CRISPR nuclease.

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides aspublished in Fonfara et al., “Phylogeny of Cas9 determines functionalexchangeability of dual-RNA and Cas9 among orthologous type IICRISPR-Cas systems,” Nucleic Acids Research, 2014, 42: 2577-2590. TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. Fonfara et al. also provides PAMsequences for the Cas9 polypeptides from various species.

Zinc Finger Nucleases

In some embodiments, a zinc finger nuclease (ZFN) is used to introduce agene edit into a cell. ZFNs are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease FokI. Because FokI functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active FokI dimer to form. Upondimerization of the FokI domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers. In most applications of ZFNs, proteins of4-6 fingers are used, recognizing 12-18 bp respectively. Hence, a pairof ZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the FokI domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese FokI variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci, 1999 96(6):2758-63; DreierB et al., J Mol Biol., 2000, 303(4):489-502; Liu Q et al., J Biol Chem.,2002, 277(6):3850-6; Dreier et al., J Biol Chem., 2005,280(42):35588-97; and Dreier et al., J Biol Chem. 2001,276(31):29466-78.

Transcription Activator-Like Effector Nucleases (TALENs)

In some embodiments, TALENs are utilized to introduce a gene edit into acell. TALENs represent another format of modular nucleases whereby, aswith ZFNs, an engineered DNA binding domain is linked to the FokInuclease domain, and a pair of TALENs operate in tandem to achievetargeted DNA cleavage. The major difference from ZFNs is the nature ofthe DNA binding domain and the associated target DNA sequencerecognition properties. The TALEN DNA binding domain derives from TALEproteins, which were originally described in the plant bacterialpathogen Xanthomonas sp. TALEs are comprised of tandem arrays of 33-35amino acid repeats, with each repeat recognizing a single base pair inthe target DNA sequence that is typically up to 20 bp in length, givinga total target sequence length of up to 40 bp. Nucleotide specificity ofeach repeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefitted fromthe use of obligate heterodimer variants of the FokI domain to reduceoff-target activity.

Additional variants of the FokI domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive FokI domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9 or CRISPR/Cpf1“nickase” mutants in which one of the Cas9 cleavage domains has beendeactivated. DNA nicks can be used to drive genome editing by HDR, butat lower efficiency than with a DSB. The main benefit is that off-targetnicks are quickly and accurately repaired, unlike the DSB, which isprone to NHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science,2009 326(5959):1509-12; Mak et al., Science, 2012, 335(6069):716-9; andMoscou et al., Science, 2009, 326(5959):1501. The use of TALENs based onthe “Golden Gate” platform, or cloning scheme, has been described bymultiple groups; see, e.g., Cermak et al., Nucleic Acids Res., 2011,39(12):e82; Li et al., Nucleic Acids Res., 2011, 39(14):6315-25; Weberet al., PLoS One., 2011, 6(2):e16765; Wang et al., J Genet Genomics,2014, 41(6):339-47; and Cermak T et al., Methods Mol Biol., 20151239:133-59.

Homing Endonucleases

In some embodiments, a homing endonuclease (HE) is used to introduce agene edit into a cell. HEs are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingGIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK, and Vsr-like that are derivedfrom a broad range of hosts, including eukarya, protists, bacteria,archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can beused to create a DSB at a target locus as the initial step in genomeediting. In addition, some natural and engineered HEs cut only a singlestrand of DNA, thereby functioning as site-specific nickases. The largetarget sequence of HEs and the specificity that they offer have madethem attractive candidates to create site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology, 2014, 24(8):663-80; Belfort andBonocora, Methods Mol Biol., 2014, 1123:1-26; and Hafez and Hausner,Genome, 2012, 55(8):553-69.

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., Nucleic Acids Res.,2014, 42: 2591-2601; Kleinstiver et al., G3, 2014, 4:1155-65; andBoissel and Scharenberg, Methods Mol. Biol., 2015, 1239: 171-96.

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-TevI (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., Nucleic AcidsRes., 2014, 42, 8816-29. It is anticipated that other combinations ofexisting nuclease-based approaches will evolve and be useful inachieving the targeted genome modifications described herein.

dCas9-FokI or dCpf1-Fok1 and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a FokI domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech, 2014, 32:569-76; and Guilinger et al., Nature Biotech., 2014, 32: 577-82. BecauseFokI must dimerize to become catalytically active, two guide RNAs arerequired to tether two FokI fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-TevI, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-TevI, with the expectation that off-targetcleavage can be further reduced.

RNA-Guided Endonucleases

The RNA-guided endonuclease systems as used herein can comprise an aminoacid sequence having at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% amino acid sequence identity to a wild-type exemplaryendonuclease, e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence IDNo. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282(2011). The endonuclease can comprise at least 70, 75, 80, 85, 90, 95,97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S.pyogenes, supra) over 10 contiguous amino acids. The endonuclease cancomprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids. The endonuclease can comprise at least: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease(e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in aHNH nuclease domain of the endonuclease. The endonuclease can compriseat most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typeendonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a HNH nuclease domain of the endonuclease. Theendonuclease can comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids in a RuvC nuclease domain of theendonuclease. The endonuclease can comprise at most: 70, 75, 80, 85, 90,95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9from S. pyogenes, supra) over 10 contiguous amino acids in a RuvCnuclease domain of the endonuclease.

The endonuclease can comprise a modified form of a wild-type exemplaryendonuclease. The modified form of the wild-type exemplary endonucleasecan comprise a mutation that reduces the nucleic acid-cleaving activityof the endonuclease. The modified form of the wild-type exemplaryendonuclease can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary endonuclease (e.g.,Cas9 from S. pyogenes, supra). The modified form of the endonuclease canhave no substantial nucleic acid-cleaving activity. When an endonucleaseis a modified form that has no substantial nucleic acid-cleavingactivity, it is referred to herein as “enzymatically inactive.”

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagine, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

Guide RNAs

In some embodiments, a guide RNA (gRNA) that can direct the activitiesof an associated endonuclease to a specific target site within apolynucleotide is used to introduce a gene edit into a cell. A guide RNAcan comprise at least a spacer sequence that hybridizes to a targetnucleic acid sequence of interest, and a CRISPR repeat sequence. InCRISPR Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the CRISPR Type II guide RNA (gRNA), the CRISPRrepeat sequence and tracrRNA sequence hybridize to each other to form aduplex. In CRISPR Type V systems, the gRNA comprises a crRNA that formsa duplex. In some embodiments, a gRNA can bind an endonuclease, suchthat the gRNA and endonuclease form a complex. The gRNA can providetarget specificity to the complex by virtue of its association with theendonuclease. The genome-targeting nucleic acid thus can direct theactivity of the endonuclease.

Exemplary guide RNAs include a spacer sequences that comprises 15-200nucleotides wherein the gRNA targets a genome location based on theGRCh38 human genome assembly. As is understood by the person of ordinaryskill in the art, each gRNA can be designed to include a spacer sequencecomplementary to its genomic target site or region. See Jinek et al.,Science, 2012, 337, 816-821 and Deltcheva et al., Nature, 2011, 471,602-607.

The gRNA can be a double-molecule guide RNA. The gRNA can be asingle-molecule guide RNA.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) can comprise, in the 5′ to 3′direction, an optional spacer extension sequence, a spacer sequence, aminimum CRISPR repeat sequence, a single-molecule guide linker, aminimum tracrRNA sequence, a 3′ tracrRNA sequence and an optionaltracrRNA extension sequence. The optional tracrRNA extension cancomprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

In some embodiments, a sgRNA comprises a 20 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence. In some embodiments, a sgRNAcomprises a less than a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. In some embodiments, a sgRNA comprises a more than20 nucleotide spacer sequence at the 5′ end of the sgRNA sequence. Insome embodiments, a sgRNA comprises a variable length spacer sequencewith 17-30 nucleotides at the 5′ end of the sgRNA sequence. In someembodiments, a sgRNA comprises a spacer extension sequence with a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, asgRNA comprises a spacer extension sequence with a length of less than3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100nucleotides.

In some embodiments, a sgRNA comprises a spacer extension sequence thatcomprises another moiety (e.g., a stability control sequence, anendoribonuclease binding sequence, or a ribozyme). The moiety candecrease or increase the stability of a nucleic acid targeting nucleicacid. The moiety can be a transcriptional terminator segment (i.e., atranscription termination sequence). The moiety can function in aeukaryotic cell. The moiety can function in a prokaryotic cell. Themoiety can function in both eukaryotic and prokaryotic cells.Non-limiting examples of suitable moieties include: a 5′ cap (e.g., a7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

In some embodiments, a sgRNA comprises a spacer sequence that hybridizesto a sequence in a target polynucleotide. The spacer of a gRNA caninteract with a target polynucleotide in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR-endonuclease system, a spacer sequence can be designed tohybridize to a target polynucleotide that is located 5′ of a PAM of theendonuclease used in the system. The spacer may perfectly match thetarget sequence or may have mismatches. Each endonuclease, e.g., Cas9nuclease, has a particular PAM sequence that it recognizes in a targetDNA. For example, S. pyogenes Cas9 recognizes a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

A target polynucleotide sequence can comprise 20 nucleotides. The targetpolynucleotide can comprise less than 20 nucleotides. The targetpolynucleotide can comprise more than 20 nucleotides. The targetpolynucleotide can comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30 or more nucleotides. The target polynucleotide cancomprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target polynucleotide sequence can comprise 20bases immediately 5′ of the first nucleotide of the PAM.

A spacer sequence that hybridizes to a target polynucleotide can have alength of at least about 6 nucleotides (nt). The spacer sequence can beat least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 20 nucleotides. In some examples, the spacer can comprise 19nucleotides. In some examples, the spacer can comprise 18 nucleotides.In some examples, the spacer can comprise 22 nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which may bethought of as a bulge or bulges.

A tracrRNA sequence can comprise nucleotides that hybridize to a minimumCRISPR repeat sequence in a cell. A minimum tracrRNA sequence and aminimum CRISPR repeat sequence may form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to an RNA-guided endonuclease. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

In some embodiments, a tracrRNA may be a 3′ tracrRNA. In someembodiments, a 3′ tracrRNA sequence can comprise a sequence with atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequenceidentity to a reference tracrRNA sequence (e.g., a tracrRNA from S.pyogenes).

In some embodiments, a gRNA may comprise a tracrRNA extension sequence.A tracrRNA extension sequence can have a length from about 1 nucleotideto about 400 nucleotides. The tracrRNA extension sequence can have alength of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. The tracrRNAextension sequence can have a length from about 20 to about 5000 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100nucleotides. The tracrRNA extension sequence can comprise less than 10nucleotides in length. The tracrRNA extension sequence can be 10-30nucleotides in length. The tracrRNA extension sequence can be 30-70nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt.

In some embodiments, a sgRNA may comprise a linker sequence with alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused (Jinek et al., Science, 2012, 337(6096):816-821). An illustrativelinker has a length from about 3 nucleotides (nt) to about 90 nt, fromabout 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt toabout 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20nt, from about 3 nt to about 10 nt. For example, the linker can have alength from about 3 nt to about 5 nt, from about 5 nt to about 10 nt,from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, fromabout 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 ntto about 50 nt, from about 50 nt to about 60 nt, from about 60 nt toabout 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about90 nt, or from about 90 nt to about 100 nt. The linker of asingle-molecule guide nucleic acid can be between 4 and 40 nucleotides.The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.The linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used (Jinek et al., Science, 2012,337(6096):816-821), but numerous other sequences, including longersequences can likewise be used.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

In some embodiments, a sgRNA does not comprise a uracil, e.g., at the3′end of the sgRNA sequence. In some embodiments, a sgRNA does compriseone or more uracils, e.g., at the 3′end of the sgRNA sequence. In someembodiments, a sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uracils(U) at the 3′ end of the sgRNA sequence.

A sgRNA may be chemically modified. In some embodiments, a chemicallymodified gRNA is a gRNA that comprises at least one nucleotide with achemical modification, e.g., a 2′-O-methyl sugar modification. In someembodiments, a chemically modified gRNA comprises a modified nucleicacid backbone. In some embodiments, a chemically modified gRNA comprisesa 2′-O-methyl-phosphorothioate residue. In some embodiments, chemicalmodifications enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

In some embodiments, a modified gRNA may comprise a modified backbones,for example, phosphorothioates, phosphotriesters, morpholinos, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar linkages.

Morpholino-based compounds are described in Braasch and David Corey,Biochemistry, 2002, 41(14): 4503-4510; Genesis, 2001, Volume 30, Issue3; Heasman, Dev. Biol., 2002, 243: 209-214; Nasevicius et al., Nat.Genet., 2000, 26:216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000,97: 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122: 8595-8602.

In some embodiments, a modified gRNA may comprise one or moresubstituted sugar moieties, e.g., one of the following at the 2′position: OH, SH, SCH₃, F, OCN, OCH₃, OCH₃ O(CH₂)_(n) CH₃, O(CH₂)_(n)NH₂, or O(CH₂)_(n) CH₃, where n is from 1 to about 10; C1 to C10 loweralkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl;Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; 2′-O-(2-methoxyethyl);2′-methoxy (2′-O—CH₃); 2′-propoxy (2′-OCH₂ CH₂CH₃); and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on thegRNA, particularly the 3′ position of the sugar on the 3′ terminalnucleotide and the 5′ position of 5′ terminal nucleotide. In someexamples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups.

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, pp75-77, 1980; Gebeyehu et al., Nucl. Acids Res. 1997,15:4513. A “universal” base known in the art, e.g., inosine, can also beincluded. 5-Me-C substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are aspects of base substitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Complexes of a Genome-Targeting Nucleic Acid and an Endonuclease

A gRNA interacts with an endonuclease (e.g., a RNA-guided nuclease suchas Cas9), thereby forming a complex. The gRNA guides the endonuclease toa target polynucleotide.

The endonuclease and gRNA can each be administered separately to a cellor a subject. In some embodiments, the endonuclease can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a subject. Such pre-complexed material is known as aribonucleoprotein particle (RNP). The endonuclease in the RNP can be,for example, a Cas9 endonuclease or a Cpf1 endonuclease. Theendonuclease can be flanked at the N-terminus, the C-terminus, or boththe N-terminus and C-terminus by one or more nuclear localizationsignals (NLSs). For example, a Cas9 endonuclease can be flanked by twoNLSs, one NLS located at the N-terminus and the second NLS located atthe C-terminus. The NLS can be any NLS known in the art, such as a SV40NLS. The weight ratio of genome-targeting nucleic acid to endonucleasein the RNP can be 1:1. For example, the weight ratio of sgRNA to Cas9endonuclease in the RNP can be 1:1.

Base Editing

In some embodiments, a gene is edited in a cell using base editing. BaseEditing is a technique enabling the conversion of one nucleotide intoanother without double-stranded breaks in the DNA. Base editing allowsfor conversion of a C to T, G to A, or vice versa. An example editor forcytosine includes rAPOBEC1 which is fused to a catalytically inactiveform of Cas9. The Cas9 helps to bind a site of interest and the rAPOBEC1cytidine deaminase induces the point mutation. Conversion of adeninerequires a mutant transfer RNA adenosine deaminase (TadA), a Cas9nikase, and a sgRNA. The construct is able to introduce thesite-specific mutation without introducing a strand break. In someembodiments, Base Editing is used to introduce one or more mutations ina cell described herein.

Kits

In some embodiments, the disclosure provides kits for differentiating ofstem cells and/or progenitor cells into Natural Killer (NK) cells. Insome embodiments, the disclosure provides kits for differentiating stemcells and/or progenitor cells into HSPCs. In some embodiments, thedisclosure provides kits for differentiating HSPCs into NK cells.

In some embodiments, the kits for differentiating cells comprise mediaor components to make media and instructions for use in differentiatingcells. In some embodiments, a kit comprises a first medium, a secondmedium, a third medium, and a fourth medium, and instructions fordifferentiating HSPCs from stem cells. In some embodiments, a kitcomprises the components to make the mediums in Table 18A or 18B, Table19A or 19B, Table 20A or 20B, and Table 21A or 21B, and instructions fordifferentiating HSPCs from stem cells. In some embodiments, a kitcomprises a first medium, a second medium, a third medium, a fourthmedium, a fifth medium, a sixth medium, and a seventh medium andinstructions for differentiating natural killer cells from stem cells.

In some embodiments, a kit comprises the components to make the mediumsin Table 18A or 18B, Table 19A or 19B, Table 20A or 20B, Table 21A or21B, Table 22, Table 23A or 23B, Table 24A or 24B and Table 25A or Table25B and instructions for differentiating natural killer cells from stemcells. In some embodiments, a kit comprises the components to make themediums in Table 18A, Table 19A or 19B, Table 20A, Table 21A, Table 22,Table 23A, and Table 24A and instructions for differentiating naturalkiller cells from stem cells. In some embodiments, a kit comprises thecomponents to make the mediums in Table 18B, Table 19A or 19B, Table20B, Table 21B, Table 22, Table 23B, Table 24B and Table 25A or 25B andinstructions for differentiating natural killer cells from stem cells.

In some embodiments, the kit comprises one or more of base media and aROCK inhibitor. In some embodiments, the kit comprises a base media, aROCK inhibitor, and BMP-4. In some embodiments, the kit comprises a basemedia and BMP-4. In some embodiments, the kit comprises a base media,BMP-4, FGF2, a WNT pathway activators, and Activin A. In someembodiments, the kit comprises FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNTC-59, and an activin/nodal inhibitor. In some embodiments, kit comprisesFGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59, and an activin/nodalinhibitor, and instructions for differentiation hematopoietic stem andprogenitor cells (HSPCs) from stem cells. In some embodiments, the kitcomprises a base medium, FGF2, VEGF, TPO, SCF, IL-3, and FLT3L. In someembodiments, the kit comprises a base medium, IL-3, IL-7, FLT3L, IL-15and SCF. In some embodiments, the kit comprises a base medium, IL-7,FLT3L, IL-15 and SCF. In some embodiments, the kit comprises a basemedium, IL-7, FLT3L, IL-15, SCF and nicotinamide. In some embodiments,the kit comprises instructions for differentiating NK cells from stemcells.

In some embodiments, the kit comprises at least a ROCK inhibitor, BMP-4,FGF2, WNT pathway activators, Activin A, VEGF, TPO, SCF, IL-3, FLT3L,WNT C-59, an activin/nodal inhibitor, and instructions fordifferentiating HSPCs from stem cells. In some embodiments, the kitcomprises at least a ROCK inhibitor, BMP-4, FGF2, WNT pathwayactivators, Activin A, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59, anactivin/nodal inhibitor, IL-7, IL-15, and instructions fordifferentiating NK cells from stem cells. In some embodiments, the kitcomprises at least FGF2, VEGF, TPO, SCF, IL-3, FLT3L, IL-7, IL-15, andinstructions for differentiating NK cells from HSPCs.

Treatment Methods

Provided herein, in some embodiments, are methods for treating a diseaseor disorder using a differentiated NK cell described herein or apopulation of cells comprising differentiated NK cells described herein.Also provided are methods for treating a disease or disorder using adifferentiated stem cell or a population of cells comprisingdifferentiated stem cells as described herein (i.e. hematopoietic stemand progenitor cells). In some embodiments, the disease or disorder is acancer, an autoimmune disease, and/or an infectious disease. In someembodiments, a subject has a cancer and an autoimmune disease. In someembodiments, a subject has a cancer and an infectious disease. In someembodiments, a subject has an autoimmune disease and an infectiousdisease.

In some embodiments, a cell population comprising differentiated NKcells is used to treat an infectious disease in a subject. In someembodiments, a cell population comprising differentiated stem cells isused to treat an infectious disease in a subject. In some embodiments,differentiated NK cells are engineered to comprise a CAR that binds to apathogen, such that the NK cells are activated and targets the pathogen.In some embodiments, a cell population comprising differentiated NKcells is used to treat an infectious disease in a subject that isimmunocompromised. In some embodiments, a cell population comprisingdifferentiated stem cells is used to treat an infectious disease in asubject that is immunocompromised. In some embodiments, animmunocompromised subject has reduced NK cell numbers and/or functionalNK cell impairment. In some embodiments, a subject is immunocompromiseddue to a cancer and/or cancer treatment, or due to an autoimmunedisease. As used herein, the term “infectious disease” includes alldiseases which are caused by infection with viruses or pathogenicbacteria and can be infected through respiratory organ, blood or skincontact. Non-limiting examples of such infectious diseases include, butare not limited to, hepatitis B, hepatitis C, human papilloma virus(HPV) infection, cytomegalovirus infection, viral respiratory disease,influenza and so on.

In some embodiments, a cell population comprising differentiated NKcells is used to treat an autoimmune disease in a subject. In someembodiments, a cell population comprising differentiated stem cells isused to treat an autoimmune disease in a subject. As used herein, theterm “autoimmune disease” refers to a class of diseases or disorders inwhich a subject's own antibodies react with host tissue or in whichimmune effector T cells are autoreactive to endogenous self-peptides andcause destruction of tissue. In some embodiments, the differentiated NKcells are engineered to express a CAR polypeptide that binds to immuneeffector T cells to prevent their activity.

In some embodiments, the differentiated NK cells described herein haveendogenous anti-cancer cell activity. In some embodiments, thedifferentiated stem cells described herein have endogenous anti-cancercell activity. In some embodiments, the differentiated NK cells comprisea CAR polypeptide, thus providing anti-cancer cell activity.

In some embodiments, a cell population comprising differentiated NKcells is used to treat a cancer. In some embodiments, a cell populationcomprising differentiated stem cells is used to treat a cancer. In someembodiments, the cancer is a leukemia. Non-limiting examples ofleukemias that may be treated as provided herein include chroniclymphocytic leukemia (CLL), non-Hodgkin lymphomas (e.g., diffuse largeB-cell lymphoma (DLBCL), high grade B-cell lymphoma, transformedfollicular lymphoma (FL), grade 3B FL, and Richter's transformation ofCLL), and acute lymphoblastic leukemia (ALL). In some embodiments,provided herein is a method of treating cancer in a subject (e.g.,human) in need thereof, comprising administering a cell populationcomprising differentiated NK cells described herein to the subject(e.g., wherein the subject has or has been diagnosed with cancer). Insome embodiments, provided herein is a method of treating a non-Hodgkinlymphoma (e.g., diffuse large B-cell lymphoma (DLBCL), high grade B-celllymphoma, transformed follicular lymphoma (FL), grade 3B FL, andRichter's transformation of CLL) in a subject (e.g., human) in needthereof, comprising administering a cell population comprisingdifferentiated NK cells described herein to the subject (e.g., whereinthe subject has or has been diagnosed with a non-Hodgkin lymphoma, or isat risk of a non-Hodgkin lymphoma). In some embodiments, provided hereinis a method of treating a non-Hodgkin lymphoma (e.g., diffuse largeB-cell lymphoma (DLBCL), high grade B-cell lymphoma, transformedfollicular lymphoma (FL), grade 3B FL, and Richter's transformation ofCLL) in a subject (e.g., human) in need thereof, comprisingadministering a cell population comprising differentiated stem cellsdescribed herein to the subject (e.g., wherein the subject has or hasbeen diagnosed with a non-Hodgkin lymphoma, or is at risk of anon-Hodgkin lymphoma). In some embodiments, the subject (e.g., a human)has (e.g., has been diagnosed with) a relapsed and/or refractorynon-Hodgkin lymphoma. In some embodiments, the subject (e.g., a human)has (e.g., has been diagnosed with) a non-relapsed or early stagenon-Hodgkin lymphoma. In some embodiments, provided herein is a methodof treating chronic lymphocytic leukemia (CLL) or acute lymphoblasticleukemia (ALL) in a subject (e.g., human) in need thereof, comprisingadministering a cell population comprising differentiated NK cellsdescribed herein to the subject (e.g., wherein the subject has or hasbeen diagnosed with CLL or ALL). In some embodiments, provided herein isa method of treating chronic lymphocytic leukemia (CLL) or acutelymphoblastic leukemia (ALL) in a subject (e.g., human) in need thereof,comprising administering a cell population comprising differentiatedstem cells described herein to the subject (e.g., wherein the subjecthas or has been diagnosed with CLL or ALL). In some embodiments, thesubject (e.g., a human) has (e.g., has been diagnosed with) a relapsedand/or refractory CLL or ALL. In some embodiments, the subject (e.g., ahuman) has (e.g., has been diagnosed with) a non-relapsed or early stageCLL or ALL. In some embodiments, the cell population is administered atany dose described herein, in particular, in a therapeutically effectiveamount. In some embodiments, a human being treated is an adult, e.g., ahuman over 18 years of age. In some embodiments, a human being treatedis under 18 years of age.

In some embodiments, the methods comprise delivering the cell populationcomprising differentiated NK cells of the present disclosure to asubject having a cancer (e.g., leukemia).

The step of administering may include the placement (e.g.,transplantation) of cells, e.g., NK cells, into a subject, by a methodor route that results in at least partial localization of the introducedcells at a desired site, such as tumor, such that a desired effect(s) isproduced. In some embodiments, the step of administering may includeplacement of differentiated stem cells, as described herein. In someembodiments, cells are administered by any appropriate route thatresults in delivery to a desired location in the subject where at leasta portion of the implanted cells or components of the cells remainviable. The period of viability of the cells after administration to asubject can be as short as a few hours, e.g., twenty-four hours, to afew days, to as long as several years, or even the life-time of thesubject, i.e., long-term engraftment. For example, in some embodiments,an effective amount of NK cell is administered via a systemic route ofadministration, such as an intraperitoneal or intravenous route. In someembodiments, an effective amount of differentiated stem cells isadministered via a systemic route of administration, such as anintraperitoneal or intravenous route.

A subject may be any subject for whom diagnosis, treatment, or therapyis desired. In some embodiments, the subject is a mammal. In someembodiments, the subject is a human.

In some embodiments, a cell population comprising NK cells beingadministered according to the methods described herein comprises geneedited cells (e.g., NK cells) differentiated from gene-edited stem cells(e.g., iPSC cells). In some embodiments, a cell population comprisingstem cells being administered according to the methods described hereincomprises gene edited cells (e.g., hematopoietic stem and progenitorcells) differentiated from gene-edited stem cells (e.g., iPSC cells).

In some embodiments, a cell population (e.g. comprising NK cells) beingadministered according to the methods described herein does not inducetoxicity in the subject, e.g., the NK cells do not induce toxicity innon-cancer cells. In some embodiments, a cell population (e.g.,comprising NK cells) being administered does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC).

In some embodiments, the subject being treated has no chronic immunesuppression.

An effective amount refers to the amount of a population of cells (e.g.,NK cells) needed to prevent or alleviate at least one or more signs orsymptoms of a medical condition (e.g., cancer), and relates to asufficient amount of a composition to provide the desired effect, e.g.,to treat a subject having a medical condition. An effective amount alsoincludes an amount sufficient to prevent or delay the development of asymptom of the disease, alter the course of a symptom of the disease(for example but not limited to, slow the progression of a symptom ofthe disease), or reverse a symptom of the disease. It is understood thatfor any given case, an appropriate effective amount can be determined byone of ordinary skill in the art using routine experimentation.

In some embodiments, the cells are derived from iPSCs. In someembodiments, the cells are expanded in culture prior to administrationto a subject in need thereof.

In some embodiments, a composition is provided comprising a plurality ofNatural Killer cells obtained by or derived by the methods describedherein. The composition (e.g., a cell composition) may be prepared as apharmaceutical composition (e.g., comprising a pharmaceuticallyacceptable carrier or excipient). A cell composition can also beemulsified or presented as a liposome composition, provided that theemulsification procedure does not adversely affect cell viability. Thecells and any other active ingredient can be mixed with excipients thatare pharmaceutically acceptable and compatible with the activeingredient, and in amounts suitable for use in the therapeutic methodsdescribed herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition andcan be determined by standard clinical techniques.

Modes of administration include but are not limited to injection andinfusion. In some embodiments, injection includes, without limitation,intravenous, intrathecal, intraperitoneal, intraspinal,intracerebrospinal, and intrasternal infusion. In some embodiments, theroute is intravenous. In some embodiments, cells described herein areadministered as a bolus or by continuous infusion (e.g., intravenousinfusion) over a period of time. In some embodiments, cells describedherein are administered in several doses over a period of time (e.g.,several infusions over a period of time). The cells described herein canbe administered in a single dose or in 2, 3, 4, 5, 6 or more doses (orinfusions). In some embodiments, the subject being treated is dosed(e.g., with an infusion) about every 1, 2, 3, 4, 5, 6, 7 or 8 weeks. Insome embodiments, the subject being treated is dosed (e.g., with aninfusion) every 2-4 weeks (e.g., every 2 weeks, 3 weeks or 4 weeks).

In some embodiments, cells (e.g., NK cells) are administeredsystemically, which refers to the administration of a population ofcells other than directly into a target site, tissue, or organ, suchthat it enters, instead, the subject's circulatory system and, thus, issubject to metabolism and other like processes. In some embodiments,hematopoietic stem and progenitor cells are administered systemically.

The efficacy of a treatment comprising a composition for the treatmentof a medical condition can be determined by the skilled clinician. Atreatment is considered “effective treatment,” if any one or all of thesigns or symptoms of, as but one example, levels of functional targetare altered in a beneficial manner (e.g., increased by at least 10⁰/),or other clinically accepted symptoms or markers of disease (e.g.,cancer) are improved or ameliorated. Efficacy can also be measured byfailure of a subject to worsen as assessed by hospitalization or needfor medical interventions (e.g., progression of the disease is halted orat least slowed). Methods of measuring these indicators are known tothose of skill in the art and/or described herein. Treatment includesany treatment of a disease in subject and includes: (1) inhibiting thedisease, e.g., arresting, or slowing the progression of symptoms; or (2)relieving the disease, e.g., causing regression of symptoms; and (3)preventing or reducing the likelihood of the development of symptoms.

Definitions

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value,number, frequency, percentage, dimension, size, amount, weight orlength. In some embodiments, the term “about” or “approximately” refersa range of quantity, level, value, number, frequency, percentage,dimension, size, amount, weight or length ±15%, ±10%, 9%, ±8%, ±7%, ±6%,±5%, ±4%, ±3%, ±2%, or 1% about a reference quantity, level, value,number, frequency, percentage, dimension, size, amount, weight orlength.

The term “aggregate” or “cell aggregates”, as used herein, refers to agroup of at least two cells being in physical contact with one anotherand forming a two- or three-dimensional cluster. In some embodiments, acell aggregate has a spherical shape. In some embodiments, the cellaggregate is a sphere. In some embodiments, the cell aggregate is aspheroid. The spheroid may also be referred to as a clump. In someembodiments, the cell aggregate is formed by suspension culturing.

As used herein, the term “induced pluripotent stem cells” or, iPSCs,means that the stem cells are produced from differentiated adult,neonatal or fetal cells that have been induced or changed, i.e.,reprogrammed into cells capable of differentiating into tissues of allthree germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCsproduced do not refer to cells as they are found in nature.

The term “hematopoietic stem and progenitor cells,” “hematopoietic stemcells,” “hematopoietic progenitor cells,” or “hematopoietic precursorcells” refers to cells which are committed to a hematopoietic lineagebut are capable of further hematopoietic differentiation and include,multipotent hematopoietic stem cells (hematoblasts), myeloidprogenitors, megakaryocyte progenitors, erythrocyte progenitors, andlymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) aremultipotent stem cells that give rise to all the blood cell typesincluding myeloid (monocytes and macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells),and lymphoid lineages (T cells, B cells, NK cells). The term “definitivehematopoietic stem cell” as used herein, refers to CD34+ hematopoieticcells capable of giving rise to both mature myeloid and lymphoid celltypes including T cells, NK cells and B cells. Hematopoietic cells alsoinclude various subsets of primitive hematopoietic cells that give riseto primitive erythrocytes, megakarocytes and macrophages.

As used herein, the term “NK cell” or “Natural Killer cell” refer to asubset of peripheral blood lymphocytes defined by the expression of CD56or CD16 and the absence of the T cell receptor (CD3). As used herein,the terms “adaptive NK cell” and “memory NK cell” are interchangeableand refer to a subset of NK cells that are phenotypically CD3- andCD56⁺, expressing at least one of NKG2C and CD57, and optionally, CD16,but lack expression of one or more of the following: PLZF, SYK, FceRy,and EAT-2. In some embodiments, isolated subpopulations of CD56⁺ NKcells comprise expression of CD16, NKG2C, CD57, NKG2D, NCR ligands,NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/orDNAM-1.

As used herein, the terms “disruption,” “genetic modification” or“gene-edit” generally refer to a genetic modification wherein a site orregion of genomic DNA is altered, e.g., by a deletion or insertion, byany molecular biology method, e.g., methods described herein, e.g., bydelivering to a site of genomic DNA an endonuclease and at least onegRNA. Exemplary genetic modifications include insertions, deletions,duplications, inversions, and translocations, and combinations thereof.In some embodiments, a genetic modification is a deletion. In someembodiments, a genetic modification is an insertion. In otherembodiments, a genetic modification is an insertion-deletion mutation(or indel), such that the reading frame of the target gene is shiftedleading to an altered gene product or no gene product.

As used herein, the term “engineered cell” generally refers to agenetically modified cell that is less susceptible to allogeneicrejection during a cellular transplant and/or demonstrates increasedsurvival after transplantation, relative to an unmodified cell. In someembodiments, a genetically modified cell as described herein is anengineered cell. In some embodiments, the engineered cell has increasedimmune evasion and/or cell survival compared to an unmodified cell. Insome embodiments, the engineered cell has increased cell survivalcompared to an unmodified cell. In some embodiments, the engineered cellhas (i) improved persistency, (ii) improved immune evasiveness, (iii)improved cytotoxic activity, (iv) improved ADCC activity, and/or (v)improved anti-tumor activity compared to an unmodified cell. In someembodiments, an engineered cell may be a stem cell. In some embodiments,an engineered cell may be an embryonic stem cell (ESC), an adult stemcell (ASC), an induced pluripotent stem cell (iPSC), or a hematopoieticstem or progenitor cell (HSPC). In some embodiments, an engineered cellmay be a differentiated cell. In some embodiments, an engineered cellmay be a somatic cell (e.g., immune system cells). In some embodiments,an engineered cell is administered to a subject. In some embodiments, anengineered cell is administered to a subject who has, is suspected ofhaving, or is at risk for a disease. In some embodiments, the engineeredcell is capable of being differentiated into lineage-restrictedprogenitor cells or fully differentiated somatic cells. In someembodiments, the lineage-restricted progenitor cells are pancreaticendoderm progenitors, pancreatic endocrine progenitors, mesenchymalprogenitor cells, muscle progenitor cells, blast cells, or neuralprogenitor cells. In some embodiments, the fully differentiated somaticcells are endocrine secretory cells such as pancreatic beta cells,epithelial cells, endodermal cells, macrophages, hepatocytes,adipocytes, kidney cells, blood cells, or immune system cells.

As used herein, the term “deletion” which may be used interchangeablywith the terms “genetic deletion”, “knock-out”, or “KO”, generallyrefers to a genetic modification wherein a site or region of genomic DNAis removed by any molecular biology method, e.g., methods describedherein, e.g., by delivering to a site of genomic DNA an endonuclease andat least one gRNA. Any number of nucleotides can be deleted. In someembodiments, a deletion involves the removal of at least one, at leasttwo, at least three, at least four, at least five, at least ten, atleast fifteen, at least twenty, or at least 25 nucleotides. In someembodiments, a deletion involves the removal of 10-50, 25-75, 50-100,50-200, or more than 100 nucleotides. In some embodiments, a deletioninvolves the removal of an entire target gene. In some embodiments, adeletion involves the removal of part of a target gene. In someembodiments, a deletion involves the removal of a transcriptionalregulator, e.g., a promoter region, of a target gene. In someembodiments, a deletion involves the removal of all or part of a codingregion such that the product normally expressed by the coding region isno longer expressed, is expressed as a truncated form, or expressed at areduced level. In some embodiments, a deletion leads to a decrease inexpression of a gene relative to an unmodified cell.

As used herein, the term “endonuclease” generally refers to an enzymethat cleaves phosphodiester bonds within a polynucleotide. In someembodiments, an endonuclease specifically cleaves phosphodiester bondswithin a DNA polynucleotide. In some embodiments, an endonuclease is azinc finger nuclease (ZFN), transcription activator like effectornuclease (TALEN), homing endonuclease (HE), meganuclease, MegaTAL, or aCRISPR-associated endonuclease. In some embodiments, an endonuclease isa RNA-guided endonuclease. In some embodiments, the RNA-guidedendonuclease is a CRISPR nuclease, e.g., a Type II CRISPR Cas9endonuclease or a Type V CRISPR Cpf1 endonuclease. In some embodiments,an endonuclease is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease,or a homolog thereof, a recombination of the naturally occurringmolecule thereof, a codon-optimized version thereof, or a modifiedversion thereof, or combinations thereof. In some embodiments, anendonuclease may introduce one or more single-stranded breaks (SSBs)and/or one or more double-stranded breaks (DSBs).

As used herein, the term “guide RNA” or “gRNA” generally refers to shortribonucleic acid that can interact with, e.g., bind to, to anendonuclease and bind, or hybridize to a target genomic site or region.In some embodiments, a gRNA is a single-molecule guide RNA (sgRNA). Insome embodiments, a gRNA may comprise a spacer extension region. In someembodiments, a gRNA may comprise a tracrRNA extension region. In someembodiments, a gRNA is single-stranded. In some embodiments, a gRNAcomprises naturally occurring nucleotides. In some embodiments, a gRNAis a chemically modified gRNA. In some embodiments, a chemicallymodified gRNA is a gRNA that comprises at least one nucleotide with achemical modification, e.g., a 2′-O-methyl sugar modification. In someembodiments, a chemically modified gRNA comprises a modified nucleicacid backbone. In some embodiments, a chemically modified gRNA comprisesa 2′-O-methyl-phosphorothioate residue. In some embodiments, a gRNA maybe pre-complexed with a DNA endonuclease.

As used herein, the term “insertion” which may be used interchangeablywith the terms “genetic insertion” or “knock-in”, generally refers to agenetic modification wherein a polynucleotide is introduced or addedinto a site or region of genomic DNA by any molecular biological method,e.g., methods described herein, e.g., by delivering to a site of genomicDNA an endonuclease and at least one gRNA. In some embodiments, aninsertion may occur within or near a site of genomic DNA that has beenthe site of a prior genetic modification, e.g., a deletion orinsertion-deletion mutation. In some embodiments, an insertion occurs ata site of genomic DNA that partially overlaps, completely overlaps, oris contained within a site of a prior genetic modification, e.g., adeletion or insertion-deletion mutation. In some embodiments, aninsertion involves the introduction of a polynucleotide that encodes aprotein of interest. In some embodiments, an insertion involves theintroduction of an exogenous promoter, e.g., a constitutive promoter,e.g., a CAG promoter. In some embodiments, an insertion involves theintroduction of a polynucleotide that encodes a noncoding gene. Ingeneral, a polynucleotide to be inserted is flanked by sequences (e.g.,homology arms) having substantial sequence homology with genomic DNA ator near the site of insertion.

The term “cell culture medium” as used herein refers to a solutioncontaining nutrients needed for culturing a cell. The term “cell culturemedium” as used herein may be used interchangeably with the term“culture medium,” “medium,” or “growth medium.” The medium includes acommercialized or prepared medium used in culturing the cell.

As used herein, the term “subject” refers to a mammal. In someembodiments, a subject is non-human primate or rodent. In someembodiments, a subject is a human. In some embodiments, a subject has,is suspected of having, or is at risk for, a disease or disorder. Insome embodiments, a subject has one or more symptoms of a disease ordisorder.

The term “suspension culture” or “suspension agitation”, as used herein,refers to the culture of cells, dispersed in a liquid nutrient medium.Due to this culture technique, the cells do not adhere to the solidsupport or the culture vessel. In some embodiments, in suspensionculture the culture vessel is constantly moving or agitated. It isreadily understood that suspension cultures comprise cells in variousstages of aggregation. A range of aggregate sizes are encountered in thesuspensions with sizes ranging from tens of microns in diameter (singlecells or couple of hundred aggregated cells) to aggregates hundreds ofmicrons in diameter, consisting of many thousands of cells.

As used herein the term “time sufficient to generate” a specific celltype (e.g., NK cells) may be understood as a time frame sufficient toobserve at least one differentiated cell (e.g., an NK cell) among thecell population. In some aspects, the “time sufficient to generate NKcells” includes time to achieve a plurality of NK cells in the cellpopulation (e.g., more than one NK cell). In some aspects, the “timesufficient to generate NK cells” includes time to achieve a majority ofNK cells in the cell population (e.g., time to achieve a cell populationhaving more than 50%, more than 60%, more than 70%, more than 80% ormore than 90% of NK cells).

Specific Methods of the Disclosure

Accordingly, the present disclosure relates in particular to thefollowing non-limiting compositions and methods.

In a first method, Method 1, the present disclosure provides a methodfor generating Natural Killer (NK) cells from stem cells, the methodcomprising: (a) culturing a population of stem cells in a first mediumcomprising a ROCK inhibitor under conditions sufficient to formaggregates; (b) culturing the aggregates in a second medium comprisingBMP-4; (c) culturing the aggregates in a third medium comprising BMP-4,FGF2, a WNT pathway activator, and Activin A; (d) culturing theaggregates in a fourth medium comprising FGF2, VEGF, TPO, SCF, IL-3,FLT3L, WNT C-59 and an activin/nodal inhibitor to form a cell populationcomprising hematopoietic stem and progenitor cells (HSPCs); (e)culturing the cell population in a fifth medium comprising FGF2, VEGF,TPO, SCF, IL-3 and FLT3L; (f) culturing the cell population in a sixthmedium comprising IL-3, IL-7, FLT3L, IL-15 and SCF; and (g) culturingthe cell population in a seventh medium comprising IL-7, FLT3L, IL-15and SCF for a time sufficient to generate NK cells.

In a second method, Method 2, the present disclosure provides a methodfor generating Natural Killer (NK) cells from stem cells, the methodcomprising: (a) culturing a population of stem cells in a first mediumcomprising a ROCK inhibitor under conditions sufficient to formaggregates; (b) culturing the aggregates in a second medium comprisingBMP-4; (c) culturing the aggregates in a third medium comprising BMP-4,FGF2, a WNT pathway activator, and Activin A; (d) culturing theaggregates in a fourth medium comprising FGF2, VEGF, TPO, SCF, IL-3,FLT3L, WNT C-59 and an activin/nodal inhibitor to form a cell populationcomprising hematopoietic stem and progenitor cells (HSPCs); (e)culturing the cell population in a fifth medium comprising FGF2, VEGF,TPO, SCF, IL-3 and FLT3L; (f) culturing the cell population in a sixthmedium comprising IL-3, IL-7, FLT3L, IL-15 and SCF; (g) culturing thecell population in a seventh medium comprising IL-7, FLT3L, IL-15 andSCF; and (h) culturing the cell population in an eighth mediumcomprising IL-7, FLT3L, IL-15, SCF, and, optionally, nicotinamide for atime sufficient to generate NK cells.

In another method, Method 3, the present disclosure provides a method asprovided in any one of Methods 1 or 2, wherein culturing the cellpopulation in the fifth medium in step (e) results in the cellpopulation comprising at least about 25% of HSPCs, optionally comprisingabout 25% to about 55% of HSPCs.

In another method, Method 4, the present disclosure provides a method asprovided in any one of Methods 1 to 3, wherein culturing the cellpopulation in the fifth medium in step (e) results in the cellpopulation comprising about 29% to about 50% of HSPCs.

In another method, Method 5, the present disclosure provides a method asprovided in any one of Methods 3 or 4, wherein culturing the cellpopulation in the fifth medium in step (e) results in the cellpopulation comprising about 36% of HSPCs or about 50% of HSPCs.

In another method, Method 6, the present disclosure provides a method asprovided in any one of Methods 1 to 5, wherein culturing the cellpopulation in the sixth medium in step (f) results in the formation ofprogenitor cell population comprising common lymphoid progenitor (CLP)cells.

In another method, Method 7, the present disclosure provides a method asprovided in Method 6, wherein the progenitor cell population comprisesat least about 15% of CLP cells, optionally wherein the CLP cellsexpress CD7 and CD45.

In another method, Method 8, the present disclosure provides a method asprovided in any one of Methods 6 or 7, wherein the progenitor cellpopulation comprises about 15% to about 50% of CLP cells, optionallyabout 19% to about 45%.

In another method, Method 9, the present disclosure provides a method asprovided in any one of Methods 6 to 8, wherein the progenitor cellpopulation comprises about 35% of CLP cells.

In another method, Method 10, the present disclosure provides a methodas provided in any one of Methods 1 to 9, wherein culturing the cellpopulation in the seventh medium in step (g) or the eighth medium instep (h) results in the cell population comprising at least about 70% ofNK cells.

In another method, Method 11, the present disclosure provides a methodas provided in Method 10, wherein culturing the cell population in theseventh medium in step (g) or the eighth medium in step (h) results inthe cell population comprising at least about 95% of NK cells.

In another method, Method 12, the present disclosure provides a methodas provided in any one of Methods 1 to 11, wherein the second mediumfurther comprises a ROCK inhibitor.

In another method, Method 13, the present disclosure provides a methodas provided in any one of Methods 1 to 12, wherein the ROCK inhibitor isthiazovivin or Y27632.

In another method, Method 14, the present disclosure provides a methodas provided in any one of Methods 1 to 13, wherein the WNT pathwayactivator is CHIR-99021.

In another method, Method 15, the present disclosure provides a methodas provided in any one of Methods 1 to 14, wherein the activin/nodalinhibitor is SB-431542.

In another method, Method 16, the present disclosure provides a methodas provided in any one of Methods 1 to 15, wherein (a) comprisesculturing for 12-48 hours.

In another method, Method 17, the present disclosure provides a methodas provided in any one of Methods 1 to 16, wherein (b) comprisesculturing for up to 24 hours.

In another method, Method 18, the present disclosure provides a methodas provided in any one of Methods 1 to 17, wherein (c) comprisesculturing for 1-3 days.

In another method, Method 19, the present disclosure provides a methodas provided in any one of Methods 1 to 18, wherein (d) comprisesculturing for 1-3 days.

In another method, Method 20, the present disclosure provides a methodas provided in any one of Methods 1 to 19, wherein (e) comprisesculturing for 1-3 days.

In another method, Method 21, the present disclosure provides a methodas provided in any one of Methods 1 to 20, wherein (f) comprisesculturing for at least 6 days and up to 8 days.

In another method, Method 22, the present disclosure provides a methodas provided in any one of Methods 1 to 21, wherein (g) comprisesculturing for at least 6 days and up to 21-28 days total.

In another method, Method 23, the present disclosure provides a methodas provided in any one of Methods 2 to 22, wherein (g) comprisesculturing for up to 6 days.

In another method, Method 24, the present disclosure provides a methodas provided in any one of Methods 2 to 23, wherein (h) comprisesculturing for at least 6 days and up to 10-16 days total.

In another method, Method 25, the present disclosure provides a methodas provided in any one of Methods 1 to 23, wherein: (a) comprisesculturing for 16-20 hours; (b) comprises culturing for 6-10 hours; (c)comprises culturing for 2 days; (d) comprises culturing for 2 days; (e)comprises culturing for 2 days; (f) comprises culturing for 6-8 days;and (g) comprises culturing for 6-28 days.

In another method, Method 26, the present disclosure provides a methodas provided in any one of Methods 2 to 24, wherein: (a) comprisesculturing for 16-20 hours; (b) comprises culturing for 6-10 hours; (c)comprises culturing for 2 days; (d) comprises culturing for 2 days; (e)comprises culturing for 2 days; (f) comprises culturing for 6-8 days;(g) comprises culturing for 6 days; and (h) comprises culturing for 8-16days.

In another method, Method 27, the present disclosure provides a methodas provided in any one of Methods 1 to 26, wherein steps (a)-(g) orsteps (a)-(h) occurs between 20-42 days or steps (a)-(h) occur between23 and 40 days.

In another method, Method 28, the present disclosure provides a methodas provided in any one of Methods 1 to 26, wherein steps (a)-(g) orsteps (a)-(h) occurs in less than 20 days or steps (a)-(h) occur in lessthan 30 days.

In another method, Method 29, the present disclosure provides a methodas provided in any one of Methods 1 to 28, wherein NK cells aregenerated in about 20 days or wherein NK cells are generated in about 23to 30 days.

In another method, Method 30, the present disclosure provides a methodas provided in Method 29, wherein steps (a)-(g) occurs in about 20 daysand culturing the cell population in the seventh medium in step (g)results in the cell population comprising at least about 70% NK cells or95% NK cells or steps (a)-(h) occurs in about 28 days and culturing thecell population in the eighth medium in step (h) results in the cellpopulation comprising at least about 70% NK cells or 95% NK cells orsteps (a)-(h) occurs in about 30 days and culturing the cell populationin the eighth medium in step (h) results in the cell populationcomprising at least about 70% NK cells or 95% NK cells.

In another method, Method 31, the present disclosure provides a methodas provided in any one of Methods 1 to 30, wherein the method is carriedout under suspension agitation.

In another method, Method 32, the present disclosure provides a methodas provided in Method 31, wherein suspension agitation comprisesrotation, optionally wherein the rotation speed is at least about 35 RPMto about 100 RPM.

In another method, Method 33, the present disclosure provides a methodas provided in any one of Methods 1 to 31, wherein the first and secondmedia comprises StemFlex or StemBrew medium. In another method, Method33B, the present disclosure provides a method as provided in any one ofMethods 1 to 31, wherein the first media comprises StemFlex or StemBrewmedium.

In another method, Method 34, the present disclosure provides a methodas provided in any one of Methods 1 to 32, wherein the third, fourth andfifth media comprise APEL medium. In another method, Method 34B, thepresent disclosure provides a method as provided in any one of Methods 1to 32, wherein the second, third, fourth and fifth media comprise APELmedium.

In another method, Method 35, the present disclosure provides a methodas provided in any one of Methods 1 to 33, wherein the sixth and seventhmedia comprise DMEM/F12 medium, or optionally DMEM (high glucose)/F12medium.

In another method, Method 36, the present disclosure provides a methodas provided in any one of Methods 1 to 34, wherein the sixth and seventhmedia comprise human serum, zinc sulfate, ethanolamine,β-mercaptoethanol, glucose, or any combination thereof.

In another method, Method 37, the present disclosure provides a methodas provided in any one of Methods 1 to 35, wherein the concentration ofhuman serum is about 5%-40%, the concentration of zinc sulfate is about1.7-40 mM, the concentration of ethanolamine is about 20-60 mM, theconcentration of β-mercaptoethanol is about 0-45 mM, and theconcentration of glucose is about 2-40 mM, or any combination thereof.

In another method, Method 38, the present disclosure provides a methodas provided in Method 36, wherein the concentration of β-mercaptoethanolis about 1 mM.

In another method, Method 39, the present disclosure provides a methodas provided in Method 37, wherein the sixth and seventh medium do notcomprise β-mercaptoethanol.

In another method, Method 40, the present disclosure provides a methodas provided in Method 36, wherein: (a) the concentration of human serumis about 15%, the concentration of zinc sulfate is about 37 mM, theconcentration of ethanolamine is about 50 mM, the concentration ofβ-mercaptoethanol is about 1 mM, and the concentration of glucose isabout 27 mM; or (b) the concentration of human serum is about 20%, theconcentration of zinc sulfate is about 36.2 mM, the concentration ofethanolamine is about 50 mM, and the concentration of glucose is about20 mM.

In another method, Method 41, the present disclosure provides a methodas provided in Method 37, wherein the sixth and seventh media comprisesDMEM/F12 medium, or optionally DMEM (high glucose)/F12 medium, and asupplement of human serum, zinc sulfate, ethanolamine,β-mercaptoethanol, glucose, or any combination thereof.

In another method, Method 42, the present disclosure provides a methodas provided in any one of Methods 1 to 40, wherein the supplementprovides an additional concentration of human serum of about 5%-40%, anadditional concentration of zinc sulfate of about 1.7-40 mM, anadditional concentration of ethanolamine of about 20-60 mM, anadditional concentration of β-mercaptoethanol of about 0.5-45 mM, anadditional concentration of glucose of about 2-40 mM or any combinationthereof.

In another method, Method 43, the present disclosure provides a methodas provided in Method 41, wherein (a) the additional concentration ofhuman serum is about 15%, the additional concentration of zinc sulfateis about 37 mM, the additional concentration of ethanolamine is about 50mM, the additional concentration of β-mercaptoethanol is about 1 mM, andthe additional concentration of glucose is about 10.25 mM; or (b) theadditional concentration of human serum is about 20%, the additionalconcentration of zinc sulfate is about 36.2 mM, the additionalconcentration of ethanolamine is about 50 mM, and the additionalconcentration of glucose is about 4.66 mM.

In another method, Method 44, the present disclosure provides a methodas provided in Method 42, wherein the supplement does not compriseβ-mercaptoethanol.

In another method, Method 45, the present disclosure provides a methodas provided in any one of Methods 41 to 43, wherein the eight mediumcomprises DMEM/F12 medium, or optionally DMEM (high glucose)/F12 medium.

In another method, Method 46, the present disclosure provides a methodas provided in any one of Methods 2 to 44, wherein the eight mediumcomprises human serum, zinc sulfate, ethanolamine, glucose, or anycombination thereof.

In another method, Method 47, the present disclosure provides a methodas provided in Method 46, wherein the concentration of human serum isabout 5/6-40%, the concentration of zinc sulfate is about 1.7-40 mM, theconcentration of ethanolamine is about 20-60 mM, and the concentrationof glucose is about 2-40 mM, or any combination thereof.

In another method, Method 48, the present disclosure provides a methodas provided in Method 47, wherein: (a) the concentration of human serumis about 10%, the concentration of zinc sulfate is about 37 mM, theconcentration of ethanolamine is about 50 mM, and the concentration ofglucose is about 20 mM.

In another method, Method 49, the present disclosure provides a methodas provided in any one of Methods 2 to 48, wherein the eighth mediacomprises DMEM/F12 medium, or optionally DMEM (high glucose)/F12 mediumand a supplement of human serum, zinc sulfate, ethanolamine, glucose, orany combination thereof.

In another method, Method 50, the present disclosure provides a methodas provided in Method 49, wherein the supplement provides an additionalconcentration of human serum of about 5%-40/o, an additionalconcentration of zinc sulfate of about 1.7-40 mM, an additionalconcentration of ethanolamine of about 20-60 mM, an additionalconcentration of glucose of about 2-40 mM or any combination thereof.

In another method, Method 51, the present disclosure provides a methodas provided in Method 50, wherein (a) the additional concentration ofhuman serum is about 15%, the additional concentration of zinc sulfateis about 37 mM, the additional concentration of ethanolamine is about 50mM, and the additional concentration of glucose is about 2.3 mM.

In another method, Method 52, the present disclosure provides a methodas provided in any one of Methods 1 to 51, wherein the first mediumcomprises 10 μM of the ROCK inhibitor.

In another method, Method 53, the present disclosure provides a methodas provided in any one of Methods 1 to 52, wherein the second mediumcomprises 30 ng/mL BMP-4 and, optionally, 10 μM of a ROCK inhibitor.

In another method, Method 54, the present disclosure provides a methodas provided in any one of Methods 1 to 53, wherein the third mediumcomprises 30 ng/mL BMP-4, 100 ng/mL FGF2, 3-10 μM CHIR-99021, 6 μM or 7μM CHIR-99021, and 2.5-5.0 ng/mL Activin A.

In another method, Method 55, the present disclosure provides a methodas provided in Method 54, wherein the third medium is added to thesecond medium at a 1:1 ratio.

In another method, Method 56, the present disclosure provides a methodas provided in any one of Methods 1 to 55, wherein the fourth and fifthmedia comprise 20 ng/mL FGF, 20 ng/mL VEGF, 20 ng/mL TPO, 100 ng/mL SCF,40 ng/mL IL-3, and 10-20 ng/mL FLT3L.

In another method, Method 57, the present disclosure provides a methodas provided in any one of Methods 1 to 56, wherein the fourth mediumfurther comprises 5 μM SB-431542.

In another method, Method 58, the present disclosure provides a methodas provided in any one of Methods 1 to 57,

In another method, Method 59, the present disclosure provides a methodas provided in any one of Methods 1 to 58, wherein the sixth and seventhmedia comprises 20 ng/mL IL-7, 10-20 ng/mL FLT3L, 10-20 ng/mL IL-15, and20 ng/mL SCF.

In another method, Method 60, the present disclosure provides a methodas provided in any one of Methods 1 to 59, wherein the sixth mediumcomprises 5 ng/mL IL-3.

In another method, Method 61, the present disclosure provides a methodas provided in any one of Methods 2 to 60, wherein the eighth mediumcomprises 10-20 ng/mL IL-7, 5-20 ng/mL FLT3L, 10-30 ng/mL IL-15, 20-40ng/mL SCF and, optionally, 6.5 mM nicotinamide.

In another method, Method 62, the present disclosure provides a methodas provided in Method 61, wherein the eighth medium comprises: 10 ng/mLIL-7, 7.5 ng/mL FLT3L, 15 ng/mL IL-15, 20 ng/mL SCF and, optionally, 6.5mM nicotinamide.

In another method, Method 63, the present disclosure provides a methodas provided in any one of Methods 1 to 62, wherein the HSPCs of (d)express CD34 and/or CD45.

In another method, Method 64, the present disclosure provides a methodas provided in any one of Methods 1 to 63, wherein the NK cells expressCD56 and/or CD45.

In another method, Method 65, the present disclosure provides a methodas provided in any one of Methods 1 to 64, wherein the NK cells expressat least one activating receptor.

In another method, Method 66, the present disclosure provides a methodas provided in Method 65, wherein the at least one activating receptoris selected from the group of NKp44, NKp46, NKG2D, CD16, KIR2DL4, NKp30,and any combination thereof.

In another method, Method 67, the present disclosure provides a methodas provided in any one of Methods 1 to 66, wherein the NK cells expressat least one inhibitory receptor.

In another method, Method 68, the present disclosure provides a methodas provided in Method 67, wherein the at least one inhibitory receptoris selected from the group of NKG2A, KIR3DL2, and any combinationthereof.

In another method, Method 69, the present disclosure provides a methodas provided in any one of Methods 1 to 68, wherein the NK cells expressat least one co-receptor.

In another method, Method 70, the present disclosure provides a methodas provided in Method 69, wherein the at least one co-receptor is CD94.

In another method, Method 71, the present disclosure provides a methodas provided in any one of Methods 1 to 70, wherein the NK cells compriseat least one function associated with endogenous NK cells.

In another method, Method 72, the present disclosure provides a methodas provided in Method 71, wherein the at least one function comprisesthe ability to induce cell lysis and cell death of a target cell.

In another method, Method 73, the present disclosure provides a methodas provided in any one of Methods 71 or 72, wherein the at least onefunction comprises degranulation.

In another method, Method 74, the present disclosure provides a methodas provided in Method 73, wherein degranulation comprises release ofperforin and granzyme B.

In another method, Method 75, the present disclosure provides a methodas provided in any one of Methods 73 or 74, wherein degranulationcomprises expression of CD107a on the cell surface of an NK cell.

In another method, Method 76, the present disclosure provides a methodas provided in any one of Methods 1 to 75, wherein the NK cells aregenerated without sorting CD34⁺ cells from the cell population.

In another method, Method 77, the present disclosure provides a methodas provided in any one of Methods 1 to 76, wherein the population ofstem cells is a population of engineered cells.

In another method, Method 78, the present disclosure provides a methodas provided in Method 77, wherein the stem cells are geneticallymodified by an RNA-guided endonuclease system.

In another method, Method 79, the present disclosure provides a methodas provided in Method 78, wherein the RNA-guided endonuclease system isa CRISPR system comprising a CRISPR nuclease and a guide RNA.

In another method, Method 80, the present disclosure provides a methodas provided in any one of Methods 1 to 79, wherein the stem cells areinduced pluripotent stem cells (iPSC), pluripotent stem cells (PSC),embryonic stem cells (ESC), or adult stem cells (ASC).

In another method, Method 81, the present disclosure provides a methodas provided in any one of Methods 1 to 80, wherein the stem cell is amammalian cell, optionally wherein the cell is a human cell.

The present disclosure also provides a population of stem cells,Population 82, obtained during or differentiated during any of themethods as provided in Methods 1 to 81.

In another Population, Population 83, the present disclosure provides apopulation as provided in Population 82, wherein the populationcomprises hematopoietic stem and progenitor cells.

The present disclosure also provides a composition, Composition 84,comprising the population of hematopoietic stem and progenitor cellsaccording to Population 83, for use as a medicament.

In another Population, Population 85, the present disclosure provides aPopulation of hematopoietic stem and progenitor cells obtained during ordifferentiated during any of the methods as provided in Methods 1 to 81for use in treating cancer.

In another Population, Population 86, the present disclosure provides aPopulation of hematopoietic stem and progenitor cells obtained during ordifferentiated during any of the methods as provided in Methods 1 to 81for use in treating an infectious disease or an autoimmune disease.

The present disclosure also provides a plurality of natural killer (NK)cells, Plurality 87, obtained by or derived from any of the methods asprovided in Methods 1 to 81.

The present disclosure also provides a composition, Composition 88,comprising the plurality of NK cells according to Plurality 87, for useas a medicament.

In another Plurality, Plurality 89, the present disclosure provides aPopulation of Natural Killer (NK) cells obtained by or derived from anyof the methods as provided in Methods 1 to 81 for use in treatingcancer.

In another Plurality, Plurality 90, the present disclosure provides aPopulation of Natural Killer (NK) cells obtained by or derived from anyof the methods as provided in Methods 1 to 81 for use in treating aninfectious disease or an autoimmune disease.

In another method, Method 91, the present disclosure provides a methodcomprising administering to a subject the plurality of NK cellsaccording to Plurality 87.

In another method, Method 92, the present disclosure provides a methodaccording to Method 91 wherein the plurality of NK cells is administeredto the subject as a pharmaceutical composition.

In another method, Method 93, the present disclosure provides a methodcomprising administering to a subject the population of stem cellsaccording to Population 82 or Population 83.

In another method, Method 94, the present disclosure provides a methodaccording to Method 93 wherein the population of stem cells isadministered to the subject as a pharmaceutical composition.

In another method, Method 95, the present disclosure the presentdisclosure provides a method according to any one of Methods 91 to 94,wherein the subject is a human who has, is suspected of having, or is atrisk for a cancer.

In another method, Method 96, the present disclosure the presentdisclosure provides a method according any one of Methods 91 to 94,wherein the subject is a human who has, is suspected of having, or is atrisk for an infectious disease or an autoimmune disease.

EXAMPLES Example 1: Cell Maintenance and Expansion

Maintenance of hiPSCs. Cells of human induced pluripotent stem cell(hiPSC) lines were maintained in STEMFLEX™ Complete media (LifeTechnologies, A3349401) with single cell passaging using ACCUTASE®(Stemcell Technologies 07920 or equivalent) on BIOLAMININ 521 LN(LN521), BIOLAMININ 511 In (ln511), or Recombinant Laminin iMatrix-511E8 (AMSBIO, AMS.892 011). For passaging, 2 μM Thiazovivin was added.Optionally, 1% REVITACELL™ Supplement was added for passaging.

Example 2: Differentiating Stem Cells into Natural Killer Cells—Protocol1

Published differentiation protocols that take 5-6 weeks to generate iNKcells (NK cells differentiated from iPSC) typically utilize spinaggregation, adherent differentiation with feeder layers, and cellsorting (see FIG. 1). As disclosed herein, a modified protocol (i.e.,Protocol 1, also called Aligned Process 1.0 or AP1.0) was developed thatis more amenable to scale-up, utilizes spontaneous aggregation, does notrequire feeder layers or cell sorting, and a shorter timeline, e.g.,about 14-28 days to generate iNK cells (see FIG. 1). Protocol 1 wasutilized to differentiate stem cells, such as wild-type and/or editedinduced pluripotent stem (iPS) cells, into hematopoietic stem andprogenitor cells (HSPCs) and then into natural killer (NK) cells. Priorto differentiation, frozen iPS cells were thawed and re-suspended inMED-A medium (Table 1). Flasks pre-coated with laminin-521 were used forcell culturing. Medium was changed daily using MED-B (Table 2) mediumuntil cells were used for differentiation.

NK Cell Differentiation. iPS cells were differentiated using thefollowing steps:

1. Day −1 (afternoon), iPSC aggregation: MED-B medium was aspirated fromflasks containing iPSC and the cells were washed with DPBS (no calcium,no magnesium) (Thermo Fisher Scientific, 14190250). DPBS was aspiratedand 2 mL ACCUTASE® (Innovative Cell Technologies, AT-104) was added perT25 flask (or 80 μL of ACCUTASE® per 1 cm²). Cells were incubated at 37°C. for 3-5 min or until all the colonies detached. Accutase digestedcells were diluted with MED-B medium to a ratio of at least 3:1(MED-B:ACCUTASE®). Cells were gently resuspended and transferred to aconical tube. Optionally, enough MED-B medium was added to cells todilute the ACCUTASE® to a ratio of 4:1 (MED-B:ACCUTASE®). Cells werepelleted and re-suspended in 10 mL of MED-C medium (Table 3). Cells werecounted and the cell concentration was diluted to 1×10⁶/mL. 6×10⁶ cellswere transferred to another tube and resuspended in a total of 6 mL ofMED-C medium. The cells were transferred to 1 well of ultra-low adhesion6-well plate (Corning, 3471) and the plate was placed on a platformshaker and rotated at 98 RPM for 18+/−2 hours (overnight).

2. At day 0, morning, at 18+/−2 hours after iPSC aggregation: The platewas rotated in a circular motion to move aggregates towards center ofthe well and aggregates were collected in a conical tube. Alternatively,all the aggregate solution mix was collected. Aggregates were allowed tosettle for 15+/−5 minutes. Cells were resuspended in MED-D medium (Table4). The cell number in aggregates was counted. The seeding density wasadjusted as needed to resuspend 3×10⁵ cells in aggregates in 2 mL MED-Dmedium and plated in one well of a 6-well low adhesion plate.Alternatively, for scale up, an appropriate number of cells wasresuspended and transferred to a PBS spinner vessel (PBS Biotech).Seeding density tested for PBS seeding vessel was approximately1-1.2×10⁵ cells per mL per final media volume (day 0+8 hrs). The platewas placed on a platform shaker and rotated at 98 RPM for 8 hours or thePBS spinner vessel were placed on a PBS base (PBS-MINI MagDrive BaseUnit; PBS Biotech), in CO₂ incubator with a rotation speed of RPM 38 to39.

3. At day 0, afternoon, at 8 hours after MED-D medium addition: 2 mL perwell of MED-E medium (Table 5) was added or scaled up for PBS spinnervessels. The plate was returned to platform shaker or PBS spinner vesselto its base in the CO2 incubator and left undisturbed until day 2. MED-Emedium components were 2× of their final concentration, therefore it wasadded to cells in MED-D at a 1:1 volume ratio.

4. At day 2: MED-E medium was replaced with MED-F medium (Table 6).

5. At day 4: MED-F medium was replaced with MED-G medium (Table 7).

6. At day 6: Starting at day 6, medium with all aggregates and singlecells was transferred into an appropriate volume centrifuge conicaltube. Cells were centrifuged and resuspended in MED-H medium (Table 8)and placed back into original wells and onto platform shaker, or intooriginal vessels and onto base, and returned for continued culture.

7. At day 10: Half or full media change was made with MED-H medium.

8. At day 14: Full media change was made with MED-I medium (Table 9).

9. From day 17 onwards: Starting at day 17 (and/or at day 20, and every2 to 3 days from day 20 onwards), single cell density was estimated fromcell culture. If cell density exceeded 3×10⁶, cells were diluted to1-2×10⁶ either by topping up cultures with fresh MED-I medium or by acomplete medium change if medium color has completely turned yellow.

TABLE 1 Medium composition for MED-A Working Stock Component Conc.Volume¹ Conc. STEMFLEX ™ Basal 90% 900 mL 100% (Thermo Fisher, A3349401)STEMFLEX ™ Supplement 1X 100 mL 10X (Thermo Fisher, A3349401)Thiazovivin 2 μM 200 μL 10 mM (Biological Industry, 1226056-71-8)¹Volumes are approximate to get the desired concentration.

TABLE 2 Medium composition for MED-B Component Working Conc. Volume¹Stock Conc. STEMFLEX ™ Basal 90% 900 mL 100% (Thermo Fisher, A3349401)STEMFLEX ™ Supplement 1X 100 mL 10X (Thermo Fisher, A3349401) ¹Volumesare approximate to get the desired concentration.

TABLE 3 Medium composition for MED-C Working Component Conc. Volume¹Stock Conc. STEMFLEX ™ Basal 90%  899 mL 100% (Thermo Fisher, A3349401)STEMFLEX ™ Supplement 1X  100 mL 10X (Thermo Fisher, A3349401)Thiazovivin 10 μM 1000 μL 10 mM (Biological Industry, 1226056-71-8)¹Volumes are approximate to get the desired concentration.

TABLE 4 Medium composition for MED-D Working Component Conc. Volume¹Stock Conc. STEMdiff APEL 2 Medium 100%  999 mL 100% (STEMCELLTechnologies, 05275) rh BMP-4 30 ng/mL  300 μL 100 μg/mL (Peprotech,120-05ET) Thiazovivin 10 μM 1000 μL  10 mM (Biological Industry,1226056-71-8) ¹Volumes are approximate to get the desired concentration

TABLE 5 Medium composition for MED-E Working Component Conc. Volume¹Stock Conc. STEMdiff APEL 2 Medium 100%  998 mL 100% (STEMCELLTechnologies, 05275) rh BMP-4  30 ng/mL  300 μL 100 μg/mL (Peprotech,120-05ET) rh FGF2 100 ng/mL 1000 μL 100 μg/mL (Peprotech, 100-18C-1MG)CHIR-99021  6 μM  600 μL  10 mM (Selleckchem, S1263) Activin-A  5 ng/mL 100 μL  50 μg/mL (R&D Systems, 338-AC-01M/CF ¹Volumes are approximateto get the desired concentration.

TABLE 6 Medium composition for MED-F Working Component Conc. Volume¹Stock Conc. STEMdiff APEL 2 Medium 100%  997 mL 100% (STEMCELLTechnologies, 05275) rh FGF2  20 ng/mL  200 μL 100 μg/mL (Peprotech,100-18C-1MG) rh VEGF165  20 ng/mL  200 μL 100 μg/mL (Peprotech,100-20-1MG) rh TPO  20 ng/mL  200 μL 100 μg/mL (Peprotech, 300-18) rhSCF 100 ng/mL 1000 μL 100 μg/mL (Peprotech, 300-07) rh IL-3  40 ng/mL 400 μL 100 μg/mL (Peprotech, 200-03-100UG) rh Flt3L  20 ng/mL  200 μL100 μg/mL (Peprotech, 300-19) WNT C-59  2 μM  200 μL  10 mM(Selleckchem, S7037) SB431542  5 μM  500 μL  10 mM (Selleckchem, S1067)¹Volumes are approximate to get the desired concentration.

TABLE 7 Medium composition for MED-G Working Component Conc. Volume¹Stock Conc. STEMdiff APEL 2 Medium 100%  998 mL 100% (STEMCELLTechnologies, 05275) rh FGF2  20 ng/mL  200 μL 100 μg/mL (Peprotech,100-18C-1MG rh VEGF165  20 ng/mL  200 μL 100 μg/mL (Peprotech,100-20-1MG) rh TPO  20 ng/mL  200 μL 100 μg/mL (Peprotech, 300-18) rhSCF 100 ng/mL 1000 μL 100 μg/mL (Peprotech, 300-07) rh IL-3  40 ng/mL 400 μL 100 μg/mL (Peprotech, 200-03-100UG) rh Flt3L  20 ng/mL  200 μL100 μg/mL (Peprotech, 300-19) ¹Volumes are approximate to get thedesired concentration.

TABLE 8 Medium composition for MED-H Working Component Conc. Volume¹Stock Conc. DMEM (high glucose, 55.47%  555 mL 100% GlutaMAX) (ThermoFisher, 10566016) F-12 with GlutaMAX 27.74%  277 mL 100% (Thermo Fisher,31765035) GlutaMAX 1X  10 mL 100X (Thermo Fisher, 35050079) Glucose*10.25 mM   4.1 mL 2500 mM Human AB serum   15%  150 mL 100% (ValleyBiomedical Inc, HP1022) Zinc sulfate   37 μM  978 μL  37 mM (MilliporeSigma, Z0251) Ethanolamine   50 μM   3 μL  16.6 M (Millipore Sigma,E0135) Ascorbic acid   20 μg/mL 2000 μL  10 mg/mL (Fisher Scientific,NC0762606) Sodium selenite    5 ng/mL  50 μL  100 μg/mL (MilliporeSigma, S9133-1MG) β-mercaptoethanol    1 μM  18 μL  55 mM (ThermoFisher, 21985-023) rh IL-3    5 ng/mL  50 μL  100 μg/mL (Peprotech,200-03-100UG) rh IL-7   20 ng/mL  200 μL  100 μg/mL (Peprotech, 200-07)rh Flt3L   15 ng/mL  150 μL  100 μg/mL (Peprotech, 300-19) rh IL-15   15ng/mL  150 μL  100 μg/mL (Peprotech, 200-15) rh SCF   20 ng/mL  200 μL 100 μg/mL (Peprotech, 300-07) *Total glucose concentration in medium is27 mM (accounting for glucose in DMEM (high glucose) medium, F12supplement and added glucose provided here). ¹Volumes are approximate toget the desired concentration.

TABLE 9 Medium composition for MED-I Working Component Conc. Volume¹Stock Conc. DMEM (high glucose, 55.47%  555 mL 100% GlutaMAX) (ThermoFisher, 10566016) F-12 with GlutaMAX 27.74%  277 mL 100% (Thermo Fisher,31765035) GlutaMAX 1X  10 mL 100X (Thermo Fisher, 35050079) Glucose*10.25 mM   4.1 mL 2500 mM Human AB serum   15%  150 mL 100% (ValleyBiomedical Inc, HP1022) Zinc sulfate   37 μM  978 μL  37 mM (MilliporeSigma, Z0251) Ethanolamine   50 μM   3 μL  16.6 M (Millipore Sigma,E0135) Ascorbic acid   20 μg/mL 2000 μL  10 mg/mL (Fisher Scientific,NC0762606) Sodium selenite    5 ng/mL  50 μL  100 μg/mL (MilliporeSigma, S9133-1MG) β-mercaptoethanol    1 μM  18 μL  55 mM (ThermoFisher, 21985-023) rh IL-7   20 ng/mL  200 μL  100 μg/mL (Peprotech,200-07) rh Flt3L   15 ng/mL  150 μL  100 μg/mL (Peprotech, 300-19) rhIL-15   15 ng/mL  150 μL  100 μg/mL (Peprotech, 200-15) rh SCF   20ng/mL  200 μL /mL (Peprotech, 300-07) *Total glucose concentration inmedium is 27 mM (accounting for glucose in DMEM (high glucose) medium,F12 supplement and added glucose provided here). ¹Volumes areapproximate to get the desired concentration.

Representative culture samples were harvested at various days for FACSand TruSeq analysis to monitor differentiation of the cells as well asto determine the morphology of the cells (see FIG. 2A). For flowcytometry analysis, live cells were collected, washed with 1% BSA in PBSor a commercial cell staining buffer (e.g. Biolegend Cat no 420201), andincubated with appropriate antibody cocktails in 5% BSA in PBS or acommercial cell staining buffer (e.g. Biolegend Cat no 420201) for atleast 20 min on ice. The cells were washed and resuspended in 1% BSA inPBS or a commercial cell staining buffer (e.g. Biolegend Cat no 420201)containing 1:1000 SyTOX Blue Dead Cell dye followed by loading the plateon the Flow cytometer for analysis (see Table 11 for antibodies used).For truseq analysis, RNA was extracted from flash frozen cell pelletsusing the Qiagen RNeasy 96 kit (Cat #74182, Qiagen) with on-column DNasetreatment (Cat #79256, Qiagen) according to the manufacturer'sinstructions, and eluted in a 50 uL volume. QC and quantification of theeluted RNA was performed using the Qiagen QIAxcel RNA QC kit v2.0 (Cat#929104, Qiagen) according to the manufacturer's instructions. Thelibrary was prepared using the Illumina TruSeq Targeted RNA Custom PanelKit, according to the manufacturer's instructions, using 32amplification cycles. QC and quantification of the DNA libraries wasperformed on the QIAxcel using the Qiagen QIAxcel DNA High ResolutionKit (Cat #929002, Qiagen) according to the manufacturer's instructions.The DNA libraries were normalized, pooled, and sequenced on the IlluminaMiseq instrument. Analysis was performed and the summed read counts werenormalized to GAPDH. Gene expression levels were expressed as foldchange vs. Day 0.

FIG. 2A shows the stage-wise differentiation of iPSC to iNK cells andvarious markers that are characteristic of the different stages duringNK differentiation. Single cells emerge at days 6-10 and iNK cellsemerge between day 10 to day 20. Cells at day 10 of the differentiatedprocess were analyzed by flow cytometry to determine expression of HSCbiomarkers CD45 and CD34 and cells at day 14 were analyzed by flowcytometry to determine expression of CLP biomarkers CD7 and CD45 (FIG.2B). The gene expression profiles indicated that NK cells develop on day20 with the loss of iPSC and HSPC marker expression. As shown in FIG.2B, HSC cells were present at day 10 of differentiation and CLP cellswere present at day 14 of differentiation consistent with the notionthat iNK cells are differentiated via HSPC and CLP stages. Cells at day6 of the differentiated process were also analyzed by flow cytometry todetermine expression of HSC biomarkers CD45 and CD34.

The differentiation process was repeated numerous times where at day 6,at least about 38-55% of the population of cells were CD34⁺ cells, whileat least about 29% to 49% of the population of cells were CD34⁺CD45⁺cells. At day 14, at least about 19-45% of the population of cells wereCD7⁺CD45⁺ cells. At day 20, at least about 70% of the population ofcells were CD56⁺CD45⁺ cells.

At days 6 and 10, early HSPC marker expression (CD34 and CD343) wasexamined (FIG. 3A). Throughout the differentiation process, cells wereanalyzed for CD45 and CD56 expression by flow cytometry (FIG. 3B),showing efficient and robust differentiation. Cell aggregates wereanalyzed at Days 10 and 14 while single cells were analyzed at Days 20,28, and 35. CD56+ cells first appeared at 2 weeks and constituted thebulk of the population after 3-4 weeks of differentiation. Intracellularpluripotency markers (Oct3/4 and SOX2) flow profiles in iPSC and day 2,4, 6 and 21 of iNK differentiation showed significant decrease of OCT3/4starting on day 2 and both markers were eliminated starting on day 4onward (FIG. 3C). The gene expression profile indicated NK celldevelopment on day 20 with loss of iPSC and HSPC markers expression(FIG. 3C and FIG. 3D and Table 10).

TABLE 10 Gene expression profile of iNK cells Log2 fold change FoldChange (normalized (normalized NK cell related markers NK cell relatedmarkers to Day 0) to Day 0) (Day 20) (Day 35) 2 to 7 ~10 to 100 EOMES,NFIL3, FCGR3A, EOMES, NFIL3, FCGR3A, fold KIR2DL1, KIR2DS4, GZMM, IL15,KLRF1 KIR2DL3, KIR3DL1, (NKP80), KLRD1 (CD94) KIR3DL2, IL15, IL18,IL2RA, KLRF1 (NKP80), SLAMF7 8 to 10 100 fold to TBX21, NCR1, NCR2,TBX21, NCR1, NCR2, 1000 fold CCR5, CD226 (DNAM-1), KIR2DL1, KIR3DL1,GZMM, IL2RB, KLRD1 KIR3DL2, IL2RA, IL2RB, (CD94) SLAMF7 >10 >1000 foldGZMA, GZMH, GZMK, GZMA, GZMH, GZMK, NCR3, CCL3, CCL4, CCL5, NCR3, CCL3,CCL4, CCL5, CCR1, IL2RG, KLRB1, CCR1, CCR5, CD226 KLRC1 (NKG2A), KLRC2(DNAM-1), IL2RG, (NKG2C) KIR2DL1, KIR2DS4, KLRB1, KLRC1 (NKG2A), KLRC2(NKG2C)

By day 28, >99% of cells are CD56⁺ with little to no CD3⁺ cells (FIG.3E) thus indicating that there was no significant T-cell contaminationfound in iNK cultures. The percent (%) of the population expressing CD14(monocyte), CD19 (B cells), and CD41/CD61 (megakaryocyte/plateletprecursors) were also significantly low, indicating minimal or nomonocyte, B cell, or megakaryocyte/platelet precursor contamination iniNK cultures (data not shown).

Flow cytometry was performed on filtered single cells on days 21, 28,and 37 to examine NK maturation marker expression levels (activatingreceptors: NKp44, NKp46, CD16, and KIR2DL4; co-receptor: CD94; inhibitorreceptors: NKG2A, and KIR3DL2) (FIG. 3F). Other markers/receptors thatare highly expressed in iNK cells: NKP30, DNAM1, OX40, C69, NG2D), andFAS (data not shown). FIG. 3F shows that the differentiated cellsexpressed a majority of maturation markers similarly detected on PB-NK(peripheral blood-NK) cells.

TABLE 11 Antibodies for marker screening Dilu- antigen fluorophorecompany catalog # tion CD16 PE-Cy7 BioLegend 360708 1:50 CD16 APC BDBioscience 561248 1:40 CD235a / APC BioLegend 349114 1:10 Glycophorin ACD34 FITC Miltenyi 130-113-178 1:25 CD34 PE BD 555822 1:10 CD38 PE-cy7eBioscience 25-0389-42 1:25 CD43 BB515 BD 564542 1:500 CD45 PE-Cy7 BD557748 1:100 CD45 BB515 BD 564585 1:100 CD56 PE Miltenyi 130-113-3071:500 CD56 BB515 BD 564488 1:25 CD56 /NCAM1 APC BD 555518 1:10 CD57PE-Cy7 BioLegend 359624 1:10 CD7 FITC BD 561604 1:10 CD94 /KLRD1 APCMiltenyi 130-098-976 1:5 CD95/Fas1 FITC BD 555673 1:10 IL-15 APCInvitrogen MA5-23627 1:10 IL-15 PE Invitrogen MA5-23561 1:10 IL-15 FITCInvitrogen MA5-23664 1:10 KIR2DL4 / APC Miltenyi 130-112-466 1:25 CD158dKIR3DL2 / PE-Vio770 Miltenyi 130-116-180 1:100 CD158e/k NKG2A /CD159aAPC Miltenyi 130-113-563 1:5 NKG2D BB515 BD 564566 1:2.5 NKp44 /CD336 PEBD 558563 1:5 NKp46 /CD335 PE-Cy7 BD 562101 1:5 Oct3/4 PE BD Bioscience560186 1:10 SOX2 Alexa 647 BD Bioscience 562139 1:10

FIG. 4 shows that the iNK persisted and expanded in the absence offeeder cells. One IPS cell could generate ˜200 to 340 iNK in 28 days.The majority of the expansion happened between days 20-28

Perforin and granzyme-B expression in cells were measured by flowcytometry at day 16 and 24, as well as day 38 differentiated cells thatwere co-incubated with K562 cells, using commercially availableantibodies. FIG. 5 shows that WT cells at day 16 of differentiation hadlow expression of perforin or granzyme-B but had higher expression atday 24. When cells were co-incubated with K562 target cells for 10 days(“Day 38 iNK”), the intensity of perforin or granzyme-B staining waseven higher. This suggests iNK were capable of continuously replenishingintracellular lytic granules content in the presence of target cells.

iNK cells were treated with Propidium Monoazide (PMA)/Ionomycin for 4hours in an NK cell activation assay. As shown in FIG. 20, after 4 hoursof treatment, there is a loss of CD16⁺ cells, an emergence of CD56′cells, and emergence of CD56⁺CD107a⁺ cells.

The cytotoxicity of day 29 derived iNK cells towards K562 cells and RPMIcells was determined using a 24-hour killing assay. K562-GFP or RPMI-GFPcells (50,000 cells per vial) were incubated with iNK effector celllines at different ratios as indicated for 24 hours. After incubation,the cells were spun, and resuspended in 175 μl media containing SyToxBlue at a 1:1000 concentration. 25 μL of countbright beads per well wereadded. The plate was read using the Flow cytometer and 100 μL volume perwell was collected for analysis. GFP-positive, SyTox Blue-negativetarget cells (live cancer cells) and countbright beads were selected andmeasured absolute events count. Total live cells were calculated asfollows:

[Total Cells=((No of live GFP-positive cells)/(Bead count for thatsample))/(Bead count per 50 μL/2).

The % of cell lysis was calculated using following formula:

% Cell lysis=(1−((Total Number of target Cells in Test Sample)/(TotalNumber of Target Cells in Control Sample))×100. The WT iNK cellsdisplayed effective cytotoxicity against K562 (FIG. 6A) and RPMI cancercell line (FIG. 6B).

Cytokines (IFNg, TNFa) and Granzyme B secretion by iNK cells weremeasured with or without adding cancer cells K562 as a target toactivate iNK for 24 hours. ProteinSimple Ella system was used formeasurements, according to the manufacturer's instructions, with thesoftware version v.3.5.2.20 of the Simple Plex Runner software, andSimple Plex Explorer software. Custom 8-plex Ella cartridges (32×8Multiplex) were provided by ProteinSimple, along with dilution bufferwhich was used to dilute each sample at a 1:2 ratio prior to loading 40μL sample per channel. As shown in FIGS. 7A-7C, the granzyme B, IFNγ,and TNFα levels in media increased in the presence of target cellsduring 24 hours of incubation, suggesting effective activation andcytotoxic activity of iNK cells in response to cancer target.

Example 3: Effect of CHIR and Activin on CD34⁺ HSPC

This Example reports the investigation of WNT signaling pathwaymolecules, such as CHIR-99021 and Activin A, to increase yields of CD34⁺cells as well as CD56⁺ cells during the later stages of iNKdifferentiation. FIG. 8 shows a schematic of the Spin EB and modifiedSpin EB protocols along with the different components added at Day 0 andDay 2 of differentiation.

FIG. 9 shows temporal modulation of WNT signaling with small moleculespromotes CD34⁺ cell induction in Day 6 aggregates. All conditions withadding Activin A, CHIR-99021 or their combinations followed by theirinhibitors (conditions 3 to 10) yielded 2-3 times higher percentage ofCD34⁺ cells compared to basic SpinEB protocol condition without addingActivin A or CHIR-99021 (conditions 1 and 2). The highest expression ofCD34+ cells was observed in Activin A and CHIR combination conditions(conditions 7-10 on the graph).

For iPSC-HSPC differentiation optimization, CD45/CD56 expression on day28 was measured (FIG. 10). The HSPC cells on day 6 were furtherdifferentiated to iNK in the StemPro-34—based differentiation mediaTable 12) in the presence of IL7, IL15, SCF. Flt3L, (IL3 was added fordays 6-14 only). FIG. 10 shows that early modulation of WNT signalingwith small molecule affected the later stage iNK induction. The highestCD56⁺ percentage was observed with adding CHIR-99021 alone (condition#5). However, it was not an optimal condition as the single cellsproduction and expansion was lower than in conditions in the presence ofActivin A molecule (data not shown). Due to low CD56⁺ cells formation onday 28, the HSPC to iNK differentiation conditions was optimized.

TABLE 12 StemPro media composition Stem Pro-34 - based media CompanyCatalog # Stock Add, mL Stem Pro-34 SFM Gibco 10640-019 500 StemPRO-34Nutrient Gibco 10641-025  13 (all) Supplement NEAA Gibco 11140-050 100x 5 L-GLUTAMINE Gibco 2503008 100x  5 β-MERCAPTOETHANOL Gibco 21985-023 0.5 Ascorbic Acid Sigma A8960 1%  2.5

Example 4: APEL Medium Formulation Testing

This Example examines the use of an APEL medium formulation (componentslisted in Table 13) instead of a commercially available modified APEL2,(Stem Cell Technologies) media with CHIR and/or Activin A. Day 6 cellswere profiled for CD34 expression (FIG. 11). In this examplecommercially available APEL2 media in combination with Activin A,CHIR-99021 followed by WNT-C59 and SB431542 showed the highest yield ofCD34+ cells on day 6 (FIG. 11) as well as single cells on day 14 onward(data not shown). This proves APEL2 as an effective media for HSPCformation. The APEL medium formulated in Table 12 promoted formation ofCD34⁺ cells although less efficiently compared to APEL2, which indicatedthat the recipe could be further optimized.

TABLE 13 APEL medium formulation Component Media IMDM   50% Ham's F12(Gibco)   50% Chemically Defined Lipid Concentrate   1% ITS-X (Gibco)  1% GlutaMAX (Gibco)  2 mM 1-thioglycerol (Sigma-Aldrich) 450 μMAscorbic acid (Sigma-Aldrich)  50 μg/mL Human albumin (CSL Behring) 0.5% Polyvinyl alcohol (PVA) 0.05%

Example 5: Stempro34 vs DMEM/F12 Medium

This Example investigates the use of various alternative base media thatcan be used for Stage II differentiation. A modified HSPC generationprotocol based on APEL medium supplemented with cytokines and addedActivin A, CHIR-99021, WNT-C59 and SB431542 showed to be very efficientfor CD34⁺ HSPC formation, however further iNK differentiation efficiencywas extremely low. The HSPC to iNK differentiation from day 6 further onwas done in StemPro-34—based media (Table 12). Hence, the HSPC to iNKmedia was optimized. In this Example, the HSPC were differentiated toHSPS until day 6 as described in Example 2 (i.e., steps 1-5). On day 6to day 21, the cells were transferred to various basal media withsupplements as indicated in Table 14. Each media was supplemented with20 ng/mL IL7, 20 ng/mL IL15, 20 ng/mL SCF, 20 ng/mL Flt3L and 5 ng/mL ofIL-3 for days 6-14 and 20 ng/mL IL7, 20 ng/mL IL15, 20 ng/mL SCF, 20ng/mL Flt3L for days 14-21 of differentiation. On day 6, the cells weretransferred to various basal media with supplements as indicated inTable 14.

Day 21 single cells were profiled for CD45⁺/CD56⁺, CD56⁺/NKG2D⁺,CD56⁺/CD94⁺, and CD56⁺/CD16⁺ expression (FIG. 12). This data showedβ-Mercaptoethanol in StemPro-based media prevented iNK differentiation(CD45⁺/CD56⁺ cells increased from around 60% to 90%—samples #1, #2 and#3). The other basal media were comparable in the efficiency of iNKinduction on day 21. The DMEM/F12 2:1 basal media was chosen due to itsprice and higher availability for scale up production.

TABLE 14 Media compositions Supplements (L-glucose (L-glu), ascorbicacid No of (AcsAc), β-Mercaptoethanol (bME), Sodium sample Selenate(NaSe), ethanolamine (EthAmine), (FIG. 12) Basal media human serum (H.Serum))  1 StemPro34 L-glu, AcsAc, NEAA, bME 55 μM  2 StemPro34 L-glu,AcsAc, NEAA, bME 1 μM  3 StemPro34 L-glu, AcsAc, NEAA, bME 0 μM  4DMEM/F12 2:1 H. Serum, L-glu, AcsAc, NaSe, EthAmine, bME 1 μM  5DMEM/F12 2:1 H. Serum, L-glu, AcsAc, NaSe, EthAmine, bME 0 μM  8 RPMI H.Serum, L-glu, AcsAc, NaSe, EthAmine, bME 0 μM  9 CMRL H. Serum, L-glu,AcsAc, NaSe, EthAmine, bME 0 μM 11 MCDB131 H. Serum, L-glu, AcsAc, NaSe,EthAmine, bME 0 μM 13 S7V1 H. Serum, L-glu, AcsAc, NaSe, EthAmine, bME 0μM 15 DMEM/F12 2:1 H. Serum, AcsAc, ITS-x 18 APEL-2

Example 6: Design of Experiment (DoE) I

This Example reports the identification of optimized concentrations forcertain components in the base media. A design of Experiment (DoE) studywas performed to determine the concentrations of zinc sulfate,P3-mercaptoethanol, human serum, and glucose that would provide the mostdifferentiation and increased number of cells at differentiation day 20.These factors were selected based on known chemical/biological effectsof them in affect general cell proliferation and differentiation.Potential positive/negative involvement of β-mercaptoethanol, zinc ion,human serum, and glucose in NK cell or lymphocyte cell proliferation,killer cell receptor function, and cytotoxicity have been previouslyreported. There were no known studies conducted on testing optimal rangeof these factors in NK cell induction from HSPC.

To determine the effects of β-mercaptoethanol on differentiation, CD45+and CD56+ expression was measured in cells cultured in Stempro34 media,DMEM/F12 media with low zinc, low human serum, and no β-mercaptoethanol(bME), or DMEM/F12 media with low zinc, low human serum, and 50 μMβ-mercaptoethanol. FIG. 13 shows the negative effect ofβ-mercaptoethanol on iNK cell induction.

FIG. 14 shows an example of the steps of the DoE experiments and Table15 lists the variables tested in the DoE and the previous reportedconcentrations. FIG. 15 shows the conditions and yields.

TABLE 15 Dose concentrations for media compositions Range of variabilityVariable Low range High range Zinc sulfate 1.7 μM 20 μMβ-mercaptoethanol 0 50 μM Human serum 2% 20% Glucose   8 mM 20 mM

Example 7: DoE II

The results from DoE I indicated the direction of effects of thedifferent variables with zinc sulfate, human serum, and glucose having apositive effect whereas β-mercaptoethanol having a negative effect. Asecond DoE experiment (DoE II) was subsequently performed to furtheroptimize the concentrations of zinc, β-mercaptoethanol, human serumalbumin, and glucose. Table 16 lists the testing range of the variablesand the resulting optimal conditions that meet all three criteria ofmaximizing CD56⁺/CD45⁺, CD45⁺, and CD56⁺/CD16+ expressing cell yieldsfrom DoE II. FIG. 16 shows the individual conditions and cell yields.FIG. 17 shows CD45, CD16, and CD56 expression of day 20 cells conductedin replicates using conditions at center points as listed in FIG. 17. Byday 20, >99% of the population are CD45+ blood cells and >90% of thepopulation are CD56⁺CD45⁺ iNKs.

TABLE 16 Dose concentrations for media compositions Previously TestingRange Optimal reported Variable Lower Limit Upper Limit conditionsconditions Glucose 25 mM  40 mM 27.1 mM 12.5 mM or  >17 mM Zinc sulfate25 μM  40 μM 37.9 μM ~1.7 μM Human serum 20% 40% 31.4% 15%β-mercaptoethanol 0 7.5 μM   1 μM 1-24 μM

Example 8: Marker Expression Comparing 15% (AP1.0) vs 31% (DOEOptimized) Human Serum Concentration

This example reports the profiling of surface markers of Day 30 iNKcells differentiated using DoE optimized protocol. The profiling wasperformed using the surface marker kit Human Cell Surface MarkerScreening Panel (Cat no 560747, BD bioscience). As shown in Table 17,Day 30 iNK cells differentiated using DoE optimized protocol expressediNK markers CD316, CD345, CD356, CD394, NKP46 and NKP44 at levelscomparable to Day 28-35 iNK differentiated using aligned protocol asseen in FIGS. 3B and 3F).

TABLE 17 iNK markers in DOE optimized cells Day 30 DOE optimized CDsurface marker surface marker % 16 5.42 45 99.93 56 97.75 94 41.86 335(NCR1, NKP46) 83.78 336 (NKP44) 66.06

Example 9: Feeder Vs Feeder-Free Culture (Rotating Spinner Vessel VsStatic Conditions)

This Example reports the comparison of utilizing rotating spinnervessels to generate suspension cells versus static conditions togenerate aggregates. iNK cells were generated in rotating spinnervessels and compared to PB-NK cultured on feeder cells and iNK cellsderived from cells grown under static conditions and expanded with orwithout feeder cells. Day 29 iNK cells were collected for killing assay(FIG. 18). For this, K562-GFP were incubated with iNK or PB-NK effectorat different ratios as indicated for 36 hours. After incubation, thecells were spun, and resuspended in 175 μl media containing SyTox Blueat a 1:1000 concentration. 25 μL of countbright beads per well wereadded. The plate was read using the Flow cytometer and 100 μL volume perwell was collected for analysis. GFP-positive, SyTox Blue-negativetarget cells (live cancer cells) and countbright beads were selected andmeasured absolute events count. Total live cells were calculated asfollows:

[Total Cells=((No of live GFP-positive cells)/(Bead count for thatsample))/(Bead count per 50 μL/2).

The % of cell lysis was calculated using following formula:

% Cell lysis=(1−((Total Number of target Cells in Test Sample)/(TotalNumber of Target Cells in Control Sample))×100. iNK cells generatedunder static condition displayed attenuated killing ability. iNK cellsexpanded on K562 feeders under static condition have the highest killingcapacity. iNK cells cultured in spinner vessel without feeders displaycomparable killing capacity as K562 feeder-expanded iNK and PB-NK. Thisexample illustrates effective differentiation of highly cytotoxic iNKcells without using feeder cells.

Example 10: Factors Affecting Expansion and Maturation: Seeding CellDensity

This Example reports the optimization of seeding cell density toincrease iNK cell expansion rates. 3.75, 5 and 10 Million starting cellpopulations (0.75×10⁶/mL, 1×10⁶/mL and 2×10⁶/mL as indicated in theFIGS. 20A, 20B, and 20C legends, respectively) each were counted andplated in 5 mL of media comprised of DMEM (Life Technologies), F12 (LifeTechnologies), human AB serum (Valley Biomedical), Glutamax, ascorbicacid, sodium selenite and ethanolamine supplemented with 20 ng/mL ofeach: IL7, 115, SCF, Flt3L with 0, 1λ, or 2× media addition. Initialstarting populations seeded at 1×10⁶/mL and 2×10⁶/mL each expanded ˜5times over 6 days. FIG. 19A shows no refreshing media, FIG. 19B showsmedia added 1 time (indicated by arrow), and FIG. 19C shows media added2 times (indicated by two arrows). As shown in FIGS. 19A-19C, celldensity modulates NK cell expansion rate. iNK cells exhausted media andslowed division at densities over 3×10⁶ cells/mL. iNK cells at low celldensity of ˜0.75 M cells/mL did not expand as efficiently, and cellnumber collapsed quickly. The optimal cell concentration for continuouscells expansion was 1-2×10⁶ cells/mL.

Example 11: Differentiating Stem Cells into Natural KillerCells—Protocol 2

It was discovered that some induced pluripotent stem (iPS) cells did notdifferentiate efficiently with Protocol 1 (FIG. 21). As shown in thisfigure, iPSC cells from two different sources were differentiated usingAP.1.0 and single cells were harvested at differentiation day 20, 23,26, and 28 for flow cytometry of CD56⁺CD45⁺ NK cells. The purity of NKcells derived from Source 2 progressively increased across time pointsbut appeared to plateau at around day 28. Thus, Protocol 2 (also calledAligned Process 2.0 or AP2.0) was developed to differentiate these iPScells into hematopoietic stem and progenitor cells (HSPCs) and then intonatural killer (NK) cells. Prior to differentiation, frozen iPS cellswere thawed and re-suspended in NK-MED-001 medium (Table 26). Flaskspre-coated with laminin-521 were used for cell culturing. Medium waschanged daily using NK-MED-002 (Table 27) medium until cells were usedfor differentiation. As described below, in some instances NK-MED-001and NK-MED-002 were prepared using StemFlex medium instead of StemBrewmedium.

NK Cell Differentiation. iPS cells were differentiated using thefollowing steps:

1. Day −1 (afternoon), iPSC aggregation: NK-MED-002 medium was aspiratedfrom flasks containing iPSC and the cells were washed with DPBS (nocalcium, no magnesium) (Thermo Fisher Scientific, 14190250). DPBS wasaspirated and 2 mL ACCUTASE® (Innovative Cell Technologies, AT-104) wasadded per T25 flask (or 80 μL of ACCUTASE® per 1 cm2). Cells wereincubated at 37° C. for 3-5 min (not more than 7 minutes). Accutasedigested cells were diluted with cold NK-MED-002 medium to a ratio of atleast 3:1 (NK-MED-002:ACCUTASE®). Cells were gently resuspended andtransferred to a conical tube. Optionally, enough NK-MED-002 medium wasadded to cells to dilute the ACCUTASE® to a ratio of at least 1:1 and upto 4:1 (NK-MED-002:ACCUTASE®). Cells were pelleted by spinning at 20-300g for 4 to 5 minutes and re-suspended in 10 mL of NK-MED-003 medium(Table 28). Cells were counted and the cell concentration was diluted to1×10⁶/mL. 6×10⁶ cells were transferred to another tube and resuspendedin a total of 6 mL of NK-MED-003 medium. The cells were transferred to 1well of ultra-low adhesion 6-well plate (Corning, 3471) and the platewas placed on a platform shaker and rotated at 98 RPM for 18+/−2 hours(overnight).

2. At day 0, morning, at 18+/−2 hours after iPSC aggregation: The platewas rotated in a circular motion to move aggregates towards center ofthe well and aggregates were collected in a conical tube. Alternatively,all the aggregate solution mix was collected. Aggregates were allowed tosettle for 15+/−5 minutes. Cells were resuspended in NK-MED-004 medium(Table 29). The cell number in aggregates was counted. The seedingdensity was adjusted as needed to resuspend 3×10⁵ cells in aggregates in2 mL NK-MED-004 medium and plated in one well of a 6-well low adhesionplate. Alternatively, for scale up, an appropriate number of cells wasresuspended and transferred to a PBS spinner vessel (PBS Biotech).Seeding density tested for PBS seeding vessel was approximately 1×10⁵cells per mL per final media volume (day 0+8 hrs). The plate was placedon a platform shaker and rotated at 98 RPM for 8 hours or the PBSspinner vessel were placed on a PBS base (PBS-MINI MagDrive Base Unit;PBS Biotech), in CO2 incubator with a rotation speed of RPM 38 to 39.

3. At day 0, afternoon, at 8 hours after NK-MED-004 medium addition: 50mL or 250 mL per well or spinner vessel, respectively, of NK-MED-005cmedium (Table 30) was added. The plate was returned to platform shakeror PBS spinner vessel to its base in the CO2 incubator and leftundisturbed until day 2. NK-MED-005c medium components were 2× of theirfinal concentration, therefore it was added to cells in NK-MED-004 at a1:1 volume ratio.

4. At day 2: NK-MED-005c medium was replaced with NK-MED-006 medium(Table 31).

5. At day 4: NK-MED-006 medium was replaced with NK-MED-007 medium(Table 32).

6. At day 6: NK-MED-007 medium was replaced with NK-MED-008b medium(Table 33), or alternatively: starting at day 6, medium with allaggregates and single cells was transferred into an appropriate volumecentrifuge conical tube. Cells were centrifuged and resuspended inNK-MED-008b medium and placed back into original wells and onto platformshaker, or into original vessels and onto base, and returned forcontinued culture.

7. At day 10: Half or full media change was made with NK-MED-008bmedium.

8. At day 14: Full media change was made with NK-MED-009b medium (Table34).

9. At day 17: One-third media change was made NK-MED-009b medium andthen a full media change was made with NK-MED-009b medium.

From day 20 onwards: Starting at day 20, single cell density wasestimated from cell culture. A full media change was made withNK-MED-010 medium (Table 35) and cell density adjusted to within 0.8 to1.5×10⁶ cells/mL. A full media change with NK-MED-010 medium andadjustment of cell density to 0.8-1.5×10⁶ cells/mL was performed every2-3 days from day 20 to 30.

TABLE 26 Medium composition for NK-MED-001 Component Working Conc.Volume¹ Stock Conc. StemBrew Basal Media 90% 980 mL 100% StemBrewSupplement 1X 20 mL 50X Thiazovivin 2 μM 200 μL 10 mM (BiologicalIndustry, 1226056-71-8) ¹Volumes are approximate to get the desiredconcentration.

TABLE 27 Medium composition for NK-MED-002 Component Working Conc.Volume¹ Stock Conc. StemBrew Basal Media 90% 980 mL 100% StemBrewSupplement 1X  20 mL 50X ¹Volumes are approximate to get the desiredconcentration.

TABLE 28 Medium composition for NK-MED-003 Component Working Conc.Volume¹ Stock Conc. StemBrew Basal 90% 979 mL 100% StemBrew Supplement1X 20 mL 50X Thiazovivin 10 μM 1000 μL 10 mM (Biological Industry,1226056-71-8) ¹Volumes are approximate to get the desired concentration.

TABLE 29 Medium composition for NK-MED-004 Working Component Conc.Volume¹ Stock Conc. STEMdiff APEL 2 Medium 100% 999 mL 100% (STEMCELLTechnologies, 05275) rh BMP-4 30 ng/mL 300 μL 100 μg/mL (Peprotech,120-05ET) Thiazovivin 10 μM 1000 μL 10 mM (Biological Industry,1226056-71-8) ¹Volumes are approximate to get the desired concentration.

TABLE 30 Medium composition for NK-MED-005c Working Component Conc.Volume¹ Stock Conc. STEMdiff APEL 2 Medium 100% 998 mL 100% (STEMCELLTechnologies, 05275) rh BMP-4 30 ng/mL 300 μL 100 μg/mL (Peprotech,120-05ET) rh FGF2 100 ng/mL 1000 μL 100 μg/mL (Peprotech, 100-18C-1MG)CHIR-99021 7 μM 700 μL 10 mM (Selleckchem, S1263) Activin-A 5 ng/mL 100μL 50 μg/mL (R&D Systems, 338-AC-01M/CF ¹Volumes are approximate to getthe desired concentration.

TABLE 31 Medium composition for NK-MED-006b Component Working Conc.Volume¹ Stock Conc. STEMdiff APEL 2 Medium 100% 997 mL 100% (STEMCELLTechnologies, 05275) rh FGF2 20 ng/mL 200 μL 100 μg/mL (Peprotech,100-18C-1MG) rh VEGF165 20 ng/mL 200 μL 100 μg/mL (Peprotech,100-20-1MG) rh TPO 20 ng/mL 200 μL 100 μg/mL (Peprotech, 300-18) rh SCF100 ng/mL 1000 μL 100 μg/mL (Peprotech, 300-07) rh IL-3 40 ng/mL 400 μL100 μg/mL (Peprotech, 200-03-100UG) rh Flt3L 20 ng/mL 200 μL 100 μg/mL(Peprotech, 300-19) SB431542 5 μM 500 μL 10 mM (Selleckchem, S1067)¹Volumes are approximate to get the desired concentration.

TABLE 32 Medium composition for NK-MED-007 Component Working Conc.Volume¹ Stock Conc. STEMdiff APEL 2 Medium 100% 998 mL 100% (STEMCELLTechnologies, 05275) rh FGF2  20 ng/mL 200 μL 100 μg/mL (Peprotech,100-18C-1MG rh VEGF165  20 ng/mL 200 μL 100 μg/mL (Peprotech,100-20-1MG) rh TPO  20 ng/mL 200 μL 100 μg/mL (Peprotech, 300-18) rh SCF100 ng/mL 1000 μL 100 μg/mL (Peprotech, 300-07) rh IL-3  40 ng/mL 400 μL100 μg/mL (Peprotech, 200-03-100UG) rh Flt3L  20 ng/mL 200 μL 100 μg/mL(Peprotech, 300-19) ¹Volumes are approximate to get the desiredconcentration.

TABLE 33 Medium composition for NK-MED-008b Working Component Conc.Volume¹ Stock Conc. DMEM (high glucose, 50.3% 503 mL 100% GlutaMAX)(Thermo Fisher, 10566016) F-12 with GlutaMAX   28% 280 mL 100% (ThermoFisher, 31765035) GlutaMAX 1X 10 mL 100X (Thermo Fisher, 35050079)Glucose* 4.66 mM 4.2 mL 1110 mM Human AB serum   20% 20 mL 100% (ValleyBiomedical Inc, HP1022) Zinc sulfate 36.2 μM 978 μL 37 mM (MilliporeSigma, Z0251) Ethanolamine 50 μM 3 μL 16.6M (Millipore Sigma, E0135)Ascorbic acid 15 μg/mL 15 μL 10 mg/mL (Fisher Scientific, NC0762606)Sodium selenite 5 ng/mL 50 μL 100 μg/mL (Millipore Sigma, S9133-1MG) rhIL-3 5 ng/mL 50 μL 100 μg/mL (Peprotech, 200-03-100UG) rh IL-7 20 ng/mL200 μL 100 μg/mL (Peprotech, 200-07) rh Flt3L 15 ng/mL 150 μL 100 μg/mL(Peprotech, 300-19) rh IL-15 15 ng/mL 150 μL 100 μg/mL (Peprotech,200-15) rh SCF 20 ng/mL 200 μL 100 μg/mL (Peprotech, 300-07) *Totalglucose concentration in medium is 20 mM (accounting for glucose in DMEM(high glucose) medium, F12 supplement and added glucose provided here).¹Volumes are approximate to get the desired concentration.

TABLE 34 Medium composition for NK-MED-009b Working Component Conc.Volume¹ Stock Conc. DMEM (high glucose, 50.3% 503 mL 100% GlutaMAX)(Thermo Fisher, 10566016) F-12 with GlutaMAX   28% 280 mL 100% (ThermoFisher, 31765035) GlutaMAX 1X 10 mL 100X (Thermo Fisher, 35050079)Glucose* 4.66 mM 4.2 mL 1110 mM Human AB serum   20% 20 mL 100% (ValleyBiomedical Inc, HP1022) Zinc sulfate 37 μM 978 μL 37 mM (MilliporeSigma, Z0251) Ethanolamine 50 μM 3 μL 16.6M (Millipore Sigma, E0135)Ascorbic acid 15 μg/mL 1500 μL 10 mg/mL (Fisher Scientific, NC0762606)Sodium selenite 5 ng/mL 50 μL 100 μg/mL (Millipore Sigma, S9133-1MG) rhIL-7 20 ng/mL 200 μL 100 μg/mL (Peprotech, 200-07) rh Flt3L 15 ng/mL 150μL 100 μg/mL (Peprotech, 300-19) rh IL-15 15 ng/mL 150 μL 100 μg/mL(Peprotech, 200-15) rh SCF 20 ng/mL 200 μL 100 μg/mL (Peprotech, 300-07)*Total glucose concentration in medium is 20 mM (accounting for glucosein DMEM (high glucose) medium, F12 supplement and added glucose providedhere). ¹Volumes are approximate to get the desired concentration.

TABLE 35 Medium composition for NK-MED-010 Component Working Conc.Volume¹ Stock Conc. DMEM (high 60.5% 605 mL 100% glucose, GlutaMAX) F-12with GlutaMAX 28% 280 mL 100% GlutaMAX 1X 10 mL 100X Glucose* 2.33 mM2.1 mL 1110 mM Human AB serum 10% 100 mL 100% Zinc sulfate 37 μM 978 μL37 mM Ethanolamine 50 μM 3 μL 16.6M Ascorbic acid 15 μg/mL 1500 μL 10mg/mL Sodium selenite 5 ng/mL 50 μL 100 μg/mL Nicotinamide 6.5 mM 6.5 mL1000 mM rh IL-7 10 ng/mL 100 μL 100 μg/mL rh Flt3L 7.5 ng/mL 75 μL 100μg/mL rh IL-15 15 ng/mL 150 μL 100 μg/mL rh SCF 20 ng/mL 200 μL 100μg/mL *Total glucose concentration in medium is 20 mM (accounting forglucose in DMEM (high glucose) medium, F12 supplement and added glucoseprovided here). ¹Volumes are approximate to get the desiredconcentration.

Example 12: DOE IV—Optimizing Stage 2 of AP2.0

This Example reports the identification of optimized concentrations forcertain components in the media used in Stage 2 of the AP2.0differentiation protocol. A design of Experiment (DoE) study wasperformed to determine the concentrations ascorbic acid,2-mercaptoethanol, human serum and glucose that would provide the mostdifferentiation and increased number of cells at differentiation day 21.

Differentiation day 6 iPSC cells (Source 2) derived from AP.1.0 wereseeded into 6-well plates and differentiated until day 21 with variouscombinations and concentrations of DoE test variables including ascorbicacid, 2-mercaptoethanol, human serum and glucose, as indicated in FIG.22. Data on cell yield, iNK purity and CD16⁺ iNK % were collected. Basicinformation on cell line and differentiation conditions for DoE IV areprovided in Table 36 below. Recommended concentrations used in stage 2iNK differentiation partially adopted in AP2.0 are provided in Table 37.Human serum concentration was up-adjusted to 20% in finalizedNK-MED-008b and NK-MED-009b media used in AP2.0.

TABLE 36 Cell Line Source 2 Stage to optimize 2 Differentiation day 6 to20 Readout Day 21 Media NK-MED-008 and 009

TABLE 37 Recommended usage bME, μM 0 Ascorbic acid, ng/mL 15 Glucose, mM20 Human serum, % 15

Example 13: DOE V—Optimizing Stage 3 of Protocol 2 (AP2.0)

This Example reports the identification of optimized concentrations forcertain components in the media used in Stage 3 of the AP2.0differentiation protocol. A design of Experiment (DoE) study wasperformed to determine the concentrations of beta-mercaptoethanol(B-Me), nicotinamide (NAM), glucose and human serum on iNK cell yield,purity (CD56⁺CD45⁺), and various activating receptor (TRAIL), exhaustionreceptor (PD1), and framework KIR receptor expression as measured at day30.

As shown in FIG. 23, Day 20 iNK cells derived from AP1.0 were harvestedand reseeded in 6-well plates to test the effect of differentcombination and concentrations of beta-mercaptoethanol (B-Me),nicotinamide (NAM), glucose and human serum on iNK cell yield, purity(CD56⁺CD45⁺), and various activating receptor (TRAIL), exhaustionreceptor (PD1), and framework KIR receptor expression. Table 38, below,summarizes the basic information for DoE V set up. Table 39 providesrecommended concentrations for each of the variables tested in DoE V.These concentrations were used to modify NK-MED-009 medium to generateNK-MED-010 medium that is being used in AP2.0

TABLE 38 Cell Line Source 1 Stage to optimize 3 Differentiation day 20to 30 Readout Day 30 Media NK-MED-009

TABLE 39 Recommended usage bME, μM 0 NAM, mM 6.5 Glucose, mM 20 HumanSerum, % 10

Example 14: Characterization and Cytotoxicity of iNK Differentiated withProtocol 2 (AP2.0)

iPSC cells were expanded in either StemFlex (Thermo Scientific) orStemMACS iPS-Brew XF, human, medium (Miltenyi) (e.g., in NK-MED-001 andNK-MED-002) and then differentiated using Protocol 2. Single cells fromiNK cultures at differentiation day 21 (FIG. 24A), 28 (FIG. 24B), and 35(FIG. 24C) were harvested for flow cytometry for the indicated markers.Inclusion and exclusion of nicotinamide (at 6.5 mM), a component inNK-MED-010 medium, was also tested for influence on iNK purity,exhaustion, and receptor expression.

The cytotoxicity of day 28 iNK cells differentiated with AP2.0 andderived from IPSC cells expanded in StemFlex or StemMACS towards K562cells or L428 cells was determined using a 24-hour killing assay.K562-GFP or L428GFP cells (50,000 cells per well) were incubated withiNK effector cell lines at different ratios as indicated for 22 hoursbefore analysis by flow cytometry on FSC/SCC, FITC (GFP) and pacificblue (cell viability) channels. Inclusion and exclusion of nicotinamide(at 6.5 mM), a component in NK-MED-010 medium, was also tested forinfluence on iNK cytotoxicity for these cancer cell lines. iNK cellsdifferentiated using Protocol 2 (AP2.0) displayed effective cytotoxicityagainst K562 (FIG. 25A) and L428 cancer cell line (FIG. 25B). Further,expansion of the precursor cells in StemFlex or StemBrew did not affectthe cytotoxic potential of the derived cells.

Day 28 iNK cultures were treated with propidium monoazide(PMA)/ionomycin (ION) for 2 or 4 hours at 37° C., 5% CO₂. The cultureswere then profiled for degranulation marker CD107a (FIG. 26A) andPerforin (FIG. 26B). Induction mimics stimulation event (e.g., increasein CD107a expression and decrease in Perforin levels).

Day 28 iNK cultures were treated with different concentrations of PMAand ionomycin for 24 hours at 37° C., 5% CO₂. Cytokine and granzymesecretion into the conditioned medium from each treatment were measuredusing Ella multianalyte assays. Cytokine secretion of IFNg and TNFa, andthe secretion of cytotoxic granzyme B were associated with the presenceof PMA and ionomycin (FIG. 27).

Example 15: Aligned Process 2.0 (AP2.0) Promoted iNK DerivationEfficiency in Spinner Vessel Format

iPSC cells were differentiated with AP2.0 using a spinner vessel format.Single cells were harvested at differentiation day 28 for flow cytometryfor CD34, CD45, CD56, or CD16. The percentage of CD45⁺ cells was about99.4% and the percentage of CD45⁺/CD56⁺ cells was about 82.6% (thepercentages of CD34⁺CD45⁺ cells and CD56⁺CD16⁺ cells was about 2% orless). Data from four independent experiments.

The iNK cultures approached purity at day 28 and expressed high level ofNK specific activating receptors NKG2D, NKP44 and NKP46 (FIG. 28). Datafrom three independent experiments.

Example 16: Aligned Process 2.0 (AP2.0) Generated Highly Pure andCytotoxic NK Population in a Bioreactor Setting

iPSC cells were differentiated with AP2.0 using a BioFlo 320 Eppendorfsystem (FIG. 29A). Four reactions were set up to investigate the effectof the initial aggregate sizes (75 μm-Epp1/2 vs 250 μm-Epp3/4) and ofpropeller direction (upflow-Epp1/3 vs downflow-Epp2/4). Cultures fromreactions Epp1 and 2, and Epp3 and 4 were combined after day 20.Aggregates and single cells were harvested at the indicateddifferentiation days to detect CD45⁺CD56⁺ cells by flow cytometry (FIG.29B). 4-hour kill assay was performed on day 28 iNK cells derived frombioreactor using K562 cancer cell lines. A dose dependent cytotoxiceffect of the bioreactor derived iNK cells was observed (FIG. 29C).

Example 17: Effect of Various Concentrations of CHIR-99021 in NK-MED-005Medium on iNK Induction

To determine the optimal concentration of CHIR-99021, differentconcentrations (2.5 μM, 3.5 μM or 4.5 μM) were added to the NK-MED-005cmedium (Table 30) to test their effect on iNK induction. Differentiationproceeded according to Protocol 2 (AP2.0) as described in Example 11.Cells were collected and analyzed using flow cytometry to detectCD45⁺CD56⁺ cells at differentiation day 20. FIG. 30 shows that thehighest proportion of CD45⁺CD56⁺ cells were collected using a moderateamount of CHIR-99021.

Example 18: Effect of WNT-C59 in NK-MED-006 Medium on iNK Induction

The effect of including WNT-C59 (2 μM) in NK-MED-006 medium during iNKinduction using the AP2.0 protocol was examined. FIG. 31A shows bloodlineage marker expression in day 20 cultures derived using AP2.0 with orwithout addition of WNT-C59 in NK-MED-006 medium. The addition ofWNT-C59 increased proportion of cells expressing non-NK lineage markers.FIG. 31B shows that addition of WNT-C50 also altered the proportion ofiNK that expressed the myeloid progenitor marker CD33, which were knownto display limited cytotoxic and cytokine secreting activities.

Example 19: Alternatives to Differentiating Stem Cells into NaturalKiller Cells—Protocol 2.5

The differentiation protocol according to Example 11 was repeated withthe following alterations, alone or in combination:

1. During the NK Cell differentiation stage, iPS cells were cultured andaggregated using a “scaled up” approach. Specifically, the NK celldifferentiation, Step 1 (Day−1 (afternoon), iPSC aggregation) step wasperformed as follows. iPSCs were grown in T175, T225, 1-cells stack or2-cell stack and digested with Accutase as previously described.Accutase digested cells were diluted 1:1 with cold NK-MED-002 medium.Cells were gently resuspended and transferred to a conical tube. Cellswere pelleted by spinning at 20-300 g for 4 to 5 minutes andre-suspended in 10 mL of NK-MED-003 medium. Cells were counted and thecell concentration was diluted to 1×10⁶/mL. 60-100×10⁶ cells weretransferred to PBS100 and resuspended in a total of 60-100 mL ofNK-MED-003 medium correspondingly. PBS vessels were placed onto PBS baseand rotated overnight at 45 RPM.

2. ROCK Inhibitor: The ROCK inhibitor used in NK-MED-003 in the previousstep, was Y-27652 (10 μM) instead of thiazovivin.

3. Nicotinamide: Nicotinamide was omitted from NK-MED-010 (used at day20 onwards).

Cells were differentiated and characterized as described in previousexamples.

1. A method for generating Natural Killer (NK) cells from stem cells,the method comprising: (a) culturing a population of stem cells in afirst medium comprising a ROCK inhibitor under conditions sufficient toform aggregates; (b) culturing the aggregates in a second mediumcomprising BMP-4; (c) culturing the aggregates in a third mediumcomprising BMP-4, FGF2, a WNT pathway activator, and Activin A; (d)culturing the aggregates in a fourth medium comprising FGF2, VEGF, TPO,SCF, IL-3, FLT3L, and an activin/nodal inhibitor to form a cellpopulation comprising hematopoietic stem and progenitor cells (HSPCs);(e) culturing the cell population in a fifth medium comprising FGF2,VEGF, TPO, SCF, IL-3 and FLT3L; (f) culturing the cell population in asixth medium comprising IL-3, IL-7, FLT3L, IL-15 and SCF; (g) culturingthe cell population in a seventh medium comprising IL-7, FLT3L, IL-15and SCF; and (h) culturing the cell population in an eighth mediumcomprising IL-7, FLT3L, IL-15, and SCF for a time sufficient to generateNK cells.
 2. The method of claim 1, wherein culturing the cellpopulation in the sixth medium in step (f) results in the formation ofprogenitor cell population comprising common lymphoid progenitor (CLP)cells.
 3. The method of claim 2, wherein the progenitor cell populationcomprises at least about 15% of CLP cells and/or wherein the CLP cellsexpress CD7 and CD45.
 4. The method of claim 1, wherein the ROCKinhibitor is thiazovivin or Y27632; the WNT pathway activator isCHIR-99021 and/or the activin/nodal inhibitor is SB-431542.
 5. Themethod of claim 1, wherein the second medium further comprises a ROCKinhibitor and/or the eighth medium further comprises nicotinamide. 6.The method of claim 1, wherein (a) comprises culturing for 12-48 hours;(b) comprises culturing for up to 24 hours; (c) comprises culturing for1-3 days; (d) comprises culturing for 1-3 days; (e) comprises culturingfor 1-3 days; (f) comprises culturing for at least 6 days and up to 8days; (g) comprises culturing for up to 6 days; (h) comprises culturingfor at least 6 days and up to 10-16 days total; or any combination of(a), (b), (c), (d), (e), (f), (g), or (h).
 7. The method of claim 6,wherein: (a) comprises culturing for 16-20 hours; (b) comprisesculturing for 6-10 hours; (c) comprises culturing for 2 days; (d)comprises culturing for 2 days; (e) comprises culturing for 2 days; (f)comprises culturing for 6-8 days; (g) comprises culturing for 6 days;and (h) comprises culturing for 8-16 days.
 8. The method of claim 1,wherein the method is carried out under suspension agitation.
 9. Themethod of claim 1, wherein the sixth, seventh and eighth media comprisehuman serum, zinc sulfate, ethanolamine, glucose, or any combinationthereof, and/or the sixth, seventh and eighth media comprise DMEM (highglucose)/F12 medium, and a supplement of human serum, zinc sulfate,ethanolamine, glucose or any combination thereof.
 10. The method ofclaim 1, wherein the third medium is added to the second medium at a 1:1ratio.
 11. The method of claim 1, wherein the fourth media comprises 20ng/mL FGF, 20 ng/mL VEGF, 20 ng/mL TPO, 100 ng/mL SCF, 40 ng/mL IL-3,10-20 ng/mL FLT3L, and 5 μM SB-431542.
 12. The method of claim 1,wherein the fifth medium comprises 20 ng/mL FGF, 20 ng/mL VEGF, 20 ng/mLTPO, 100 ng/mL SCF, 40 ng/mL IL-3, and 10-20 ng/mL FLT3L.
 13. The methodof claim 1, wherein the sixth media comprises 20 ng/mL IL-7, 10-20 ng/mLFLT3L, 10-20 ng/mL IL-15, 20 ng/mL SCF, and 5 ng/mL IL-3.
 14. The methodof claim 1, wherein the seventh medium comprises 20 ng/mL IL-7, 10-20ng/mL FLT3L, 10-20 ng/mL IL-15, and 20 ng/mL SCF.
 15. The method ofclaim 1, wherein the eighth medium comprises 10-20 ng/mL IL-7, 5-20ng/mL FLT3L, 10-30 ng/mL IL-15, and 20-40 ng/mL SCF.
 16. The method ofclaim 15, wherein the eighth medium further comprises 6.5 mMnicotinamide
 17. The method of claim 1, wherein the NK cells express (a)at least one of CD56 or CD45 and/or (b) at least one of: an activatingreceptor, an inhibitory receptor or a co-receptor.
 18. The method ofclaim 17, wherein the at least one activating receptor is selected fromthe group of NKp44, NKp46, NKG2D, CD16, KIR2DL4, NKp30, and anycombination thereof; the at least one inhibitory receptor is selectedfrom the group of NKG2A, KIR3DL2, and any combination thereof, and/orthe at least one co-receptor is CD94.
 19. The method of claim 1, whereinthe NK cells comprise at least one function associated with endogenousNK cells.
 20. The method of claim 19, wherein the at least one functioncomprises (a) the ability to induce cell lysis and cell death of atarget cell; (b) degranulation; or (c) a combination thereof.
 21. Themethod of claim 20, wherein degranulation comprises (a) release ofperforin and granzyme B; (b) expression of CD107a on the cell surface ofan NK cell; or (c) a combination thereof.
 22. The method of claim 1,wherein the NK cells are generated without sorting CD34⁺ cells from thecell population.
 23. The method of claim 1, wherein the population ofstem cells is a population of engineered cells.
 24. The method of claim23, wherein the stem cells are genetically modified by an RNA-guidedendonuclease system.
 25. The method of claim 24, wherein the RNA-guidedendonuclease system is a CRISPR system comprising a CRISPR nuclease anda guide RNA.
 26. The method of claim 1, wherein the stem cells areinduced pluripotent stem cells (iPSC), pluripotent stem cells (PSC),embryonic stem cells (ESC), or adult stem cells (ASC).
 27. The method ofclaim 26, wherein the stem cells are mammalian cells.
 28. The method ofclaim 27, wherein the mammalian cells are human cells.
 29. A pluralityof Natural Killer (NK) cells generated by the method of claim
 1. 30. Amethod comprising administering to a subject the plurality of NK cellsof claim 29.